Method of inducing memory B cell development and terminal differentiation

ABSTRACT

A method is disclosed herein for inducing differentiation of a B cell progenitor into a memory B cells and/or a plasma cell. The method includes contacting a population of cells including a mature B cell or a B cell progenitor with an effective amount of IL-21, and isolating memory B cells or plasma cells. In one embodiment, the B cell progenitor is an immature B cell. A method is also disclosed for enhancing an immune response. The method includes contacting a population of cells including a B cell progenitor with an effective amount of IL-21, and isolating memory B cells or plasma cells. The memory B cells and/or the plasma cell are then introduced into the subject to enhance the immune response. A method is also disclosed for treating a subject with a condition comprising a specific deficiency of at least one of memory B cells and plasma cells. A method is disclosed for identifying an agent with a physiological effect on one or more of a memory B cell and a plasma cell differentiation. A method is also disclosed for identifying agents that inhibit an activity of IL-21. Methods are also disclosed for inducing apoptosis of a B cell and for decreasing the number of B cells. A method is also described for producing a B cell hybridoma.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation-In-Part application and claimspriority to and benefit of PCT Application No. PCT/US/2004/039135, filedNov. 18, 2004, which claims priority to and benefit of U.S. ProvisionalApplication No. 60/523,754, filed Nov. 19, 2003. The disclosures of bothof these documents is incorporated herein by reference for all purposes.

FIELD

This application relates to the field of immunology, specifically to theuse of IL-21 to induce differentiation of immature B cells into memory Bcells and plasma cells.

BACKGROUND

Cytokines exert their respective biochemical and physiological effectsby binding to specific receptor molecules. Receptor binding thenstimulates specific signal transduction pathways (Kishimoto et al., Cell76:253-262, 1994). The specific interactions of cytokines with theirreceptors can be the primary regulators of a wide variety of cellularprocesses including activation, proliferation, and differentiation ofcells (Arai et al., Ann. Rev. Biochem. 59:783-836, 1990; Paul and Seder,Cell 76:241-251, 1994).

Interleukin-21 (IL-21) is a type I cytokine whose receptor is expressedon T, B, and NK cells. IL-21 was isolated from a cDNA library derivedfrom activated CD3 (+) T cells (Parrish-Novak et al., Nature 408 57-63,2000). The IL-21 cDNA encodes a secreted protein of 131 amino acidsprotein most closely related to IL-2 and IL-15. The IL-21 gene has beenmapped to human chromosome 4q26-q27 near the IL-2 gene.

IL-21 mRNA is expressed in activated CD4+ but not in activated CD8+ Tcells. In addition, IL-21 expression was not detected in B cells andmonocytes (Parrish-Novak et al., Nature 408:57-63, 2000). IL-21 has alsobeen shown to stimulate proliferation of naive (CD45RA+) cells, but notmemory (CD45RO+) T cells, mediated by engagement of CD3. IL-21 has alsobeen shown to stimulate the proliferation of bone marrow progenitor tocells and to enhance the expression of the NK-cell marker CD56 in thepresence of IL-15 (for review, see Horst Ibelgaufts' COPE: CytokinesOnline Pathfinder Encyclopedia, available on the internet). In vitro,IL-21 can act as a co-mitogen for anti-CD3-induced thymocyte andperipheral T cell proliferation (Parrish-Novak et al., Nature 408:57-63,2000), augment NK cell expansion and differentiation from human CD34⁺cells when cultured with IL-15 and Flt-3 ligand, and can also activateNK-cytolytic activity (Parrish-Novak et al., Nature 408:57-63, 2000;Kasaian et al., Immunity 16:559, 2002).

The IL-21 receptor has been isolated and was found to be expressed byCD23+B cells, B cell lines, a T cell leukemia line, and NK cell lines.The receptor gene has been mapped to human chromosome 16p12 (seeParrish-Novak et al., Nature 408:57-63, 2000; Ozaki et al., Proc. Natl.Acad. Sci. USA 97:11439-11444, 2000). The receptor (538 amino acids) ismost closely related to human IL-2 receptor beta chain, and contains aWSXWS motif in the extracellular region, typical of type-1 cytokinereceptors (see Ozaki et al., Proc. Natl. Acad. Sci. USA 97:11439-11444,2000; Parrish-Novak et al., Nature 408:57-63, 2000; and Nat. Rev.Immunol. 1:200-208). The common cytokine receptor gamma chain, anindispensable subunit of the functional receptor complexes for IL-2,IL-4, IL-7, IL-9, and IL-15 has been shown also to be part of the IL-21receptor complex. The functional signaling complex signals in partthrough the activation of Jak1 and Jak3 as well as Stat1, Stat3, andStat5 (see Asao et al., J. Immunol. 167:1-5, 2000; Ozaki et al., Proc.Natl. Acad. Sci. USA 97:11439-11444, 2000). However, the specificeffects of IL-21 on the differentiation and populations of B cells andthe activity of specific B cell populations have not previously beenelucidated.

SUMMARY

A method is disclosed herein for inducing differentiation of mature Bcells and B cell progenitors into memory B cells and/or plasma cells.The method involves contacting a population of cells including maturememory cells, naïve B cells and/or B cell progenitors with IL-21 and anyappropriate costimulation agent, and isolating memory B cells and/orplasma cells. In one embodiment, the B cell progenitor is an immature Bcell.

A method is also disclosed for enhancing an immune response. The methodincludes contacting a population of cells including mature memory cells,naïve B cells and/or B cell progenitors with IL-21 in the presence ofany appropriate costimulation, and isolating memory B cells and/orplasma cells. The memory B cells and/or the plasma cell are thenintroduced into a subject to enhance the immune response.

In an embodiment, the method includes isolating a population of cellscomprising mature memory cells, naïve B cells and/or B cell progenitorsfrom a subject; contacting the population of cells with a compositioncomprising IL-21 or an agonist thereof (and any appropriatecostimulation agent) ex vivo, thereby inducing differentiation of memoryB cells and/or plasma cells. The memory B cells, the plasma cells, orboth are then isolated and introduced into a subject.

A method is also disclosed for treating a subject with a conditioncomprising an immunodeficiency characterized by a specific deficiency ofmemory B cells and/or plasma cells. The method includes administeringIL-21 or an agonist thereof to the subject with such a deficiency,thereby ameliorating a sign or symptom of the deficiency.

A method is disclosed for identifying an agent with an effect on thedifferentiation of memory B cells and/or plasma cells. The methodincludes: contacting a population of B cell progenitors with aneffective amount of IL-21 and an agent of interest; and detecting aneffect of the agent on memory B cell differentiation, plasma celldifferentiation, or both.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-D are sets of digital images and graphs demonstrating thatIL-21 is pro-apoptotic for B cells, but is anti-apoptotic for T cells,particularly CD8⁺ T cells. FIG. 1A is a scatter plot of a FACS analysisshowing that IL-21 augments DNA fragmentation (measured by TUNELstaining, Roche Applied Science, IN) in B cells stimulated withanti-CD40, anti-IgM+IL-4, or LPS. Upper, middle, and lower panelscorrespond to cells treated with anti-CD40±IL-21 for 15 hours,anti-IgM+IL-4±IL-21 for 48 hours, and LPS±IL-21 for 15 hours,respectively. FIG. 1B is a set of digital images showing caspaseactivation by IL-21 as indicated by PARP cleavage. Purified B cells wereactivated with LPS or anti-CD40, ±IL-21, for 6 hours or left untreatedas controls. Clarified cell lysates (50 μg) were run on SDS gels andWestern blotted with an anti-PARP Ab (Cell Signaling Technology, MA).The uncleaved and cleaved forms of PARP are also shown in Jurkat T cellstreated with etoposide as a positive control. FIG. 1C is a set ofscatter plots of FACS data demonstrating Bcl-2 levels were equivalent inuntreated and IL-21-treated B cells. FIG. 1D is a set of graphs showingIL-21 preferentially increases survival of CD8⁺ T cells.

FIGS. 2A-C are sets of digital images and scatter plots showing theeffect of IL-21 on B cell populations. FIG. 2A is a digital image of aNorthern blot showing expression of IL-21 mRNA in IL-21 TG mice. TotalRNA (10 μg) from bone marrow or spleen from WT or IL-21 TG mice wasNorthern blotted with human IL-21 cDNA (upper panel) or control pHe7(lower panel) probes. TG line #8 showed approximately 30 fold higherIL-21 mRNA expression than the other two lines. FIG. 2B is a FACS plotdemonstrating the lower apparent numbers of mature (M), follicular (FO),and marginal zone (MZ) B cells in IL-21 TG than in WT mice based onCD21/CD23 and IgM/CD21 splenic expression patterns. T1, transitional Bcells-1; T2, transitional B cells-2; NF, newly formed B cells. FIG. 2Cis a FACS plot similar to the one shown in FIG. 2B except that cellswere from IL-21 vector or saline injected B6 WT mice. Mice were analyzedat day 7.

FIGS. 3A-C are sets of FACS plots and bar graphs demonstrating theeffect of IL-21 on B cell populations. FIG. 3A is a scatter plotobtained from a flow cytometric analysis of B cell populations.AA4.1/B220 staining of splenocytes from WT or TG mice (i and ii). CD23profiles are shown for AA4.1^(high) (iii and iv) and AA4.1^(low)splenocytes (v and vi). IgM/IgD profiles are shown for AA4.1^(high) (viiand viii) and AA4.1^(low) (ix and x) splenocytes. FIG. 3B is a plotshowing that L-21 potently decreases CD23 expression, whereas it isinduced by IL-4. Shown are purified splenic B cells stimulated with LPSor anti-CD40 that were additionally treated with medium, IL-4, or IL-21for 10 hours. The numbers are the mean fluorescent intensities of viablecells. FIG. 3C is a set of bar graphs showing the cellularity in micefollowing murine IL-21 injection. Shown are total cell numbers±S.D.(from more than 10 animals) for splenocytes as well as B cells (definedby B220) that were AA4.1^(high) (immature B cells) and AA4.1^(low)(resting mature B cells and post-switch cells).

FIGS. 4A-I are a digital images of photomicrographs.Immunohistochemistry was performed on wild type control (A, B, C),Transgenic (D, E, F), and IL-21 plasmid injected (G, H, I) mice usingantibodies to IgD/IgM (A, D, G), MAdCAM-1/IgM (B, E, H), and MARCO/IgM(C, F, I). Spleens were embedded in tissue-tek/O.C.T. compound (Sakura,Zoeterwoude, the Netherlands), frozen in liquid nitrogen, seriallysectioned, fixed in ice-cold acetone for 5 minutes, and stained for 45minutes in a humid chamber with either biotinylated anti-MAdCAM-1(Southern Biotech), rat Ab supernatant specific for IgD (clone 1126C),or purified rat Ab specific for MCA1849 (MARCO, Serotec, Raleigh, N.C.),followed by SA-conjugated or goat anti-rat conjugated Oregon Green(Molecular Probes, Eugene, Oreg.). IgM was detected with goat anti-mouseIgM Texas Red (Southern Biotechnology). Data are representative ofseveral mice examined.

FIGS. 5A-E are graphs and digital images showing the effect of IL-21 oncell viability and Ig production in IL-21 TG mice. FIG. 5A is a scatterplot showing annexin V staining of B220+mature splenic B cells from TGline #5 mice (panel i) or IL-21 vector injected mice (panel ii). FIG. 5Bis a set of plots showing reversed CD4:CD8 ratio in IL-21 TG mice (panelii versus i) and IL-21 vector-injected mice (iv versus iii). FIG. 5C isa set of bar graphs showing that serum IgG (left) and IgM (right) levels(mean±SD) in WT and two IL-21 TG lines; 4 mice were analyzed in eachgroup. FIG. 5D is a scatter plot showing increased surface IgG1⁺B cellsin IL-21 TG mice. Splenocytes from IL-21 TG mice (line #5) and WTlittermates were stained with anti-IgG 1 and analyzed by flow cytometry.FIG. 5E is a bar graph showing ovalbumin specific Ig in WT versus IL-21TG mice (line #5) immunized with 100 μg of ovalbumin/alum. Data aremean±S.D. Three mice were analyzed in each group and a representativemean±S.D. of 3 experiments is shown.

FIGS. 6A-C are graph and showing the effects of IL-21 on anti-IgMinduced B cell proliferation, death, and differentiation. FIG. 6A is abar graph showing that IL-21 potently increased proliferation of B cellsstimulated with anti-IgM+anti-CD40. Purified B cells from B6 WT micewere cultured with anti-IgM in the presence or absence of IL-21, IL-4,and anti-CD40 for 48 hours and were then pulsed with ³H-thymidine forthe last 10 hours. Results depict the average proliferative response of3 mice analyzed in a representative experiment. FIGS. 6B and 6C areplots obtained from a flow cytometric analysis of B cells cultured for48 hours as above and analyzed for expression of syndecan-1 (FIG. 6B)and surface IgG1 (FIG. 6C). Data are representative of 3 similarexperiments.

FIGS. 7A-D are graphs and digital images showing that IL-21 treatmentincreases Blimp-1 and Bcl-6 expression while diminishing Pax5. FIG. 7Ais a set of plots showing that IL-21 induces syndecan-1 expression (leftpanel), but diminishes MHC 11 and CD23 expression (middle and rightpanels, respectively) in Bcl-1 cells. FIG. 7B is a set of bar graphsshowing that IL-21 induces Blimp-1 (left panel) and Bcl-6 (middle panel)expression but decreases expression of Pax5 (right panel) mRNA, asevaluated by real-time PCR in Bcl-1 cells. The effect of the combinationof IL-2 and IL-5 on expression of each gene is also shown. FIG. 7C is adigital image showing Induction of Blimp-1 protein, as evaluated byWestern blotting in purified splenic B cells treated with thecombination of anti-IgM plus IL-21 but not with anti-IgM alone. FIG. 7Dis a digital image showing IL-21-mediated induction of Blimp-1 and Bcl-6DNA binding activities as evaluated by EMSAs. Splenic B cells wereisolated treated with anti-IgM with or without IL-21 as described in theExperimental Procedures, and then Blimp-1 and Bcl-6 DNA bindingactivities were evaluated using specific DNA probes. An antibody toBcl-6 supershifted the Bcl-6 band, whereas an antibody to Stat3, whichcan bind the same probe (Reljic et al., J. Exp. Med. 192:1841-1847,2000), did not supershift the Bcl-6 band.

FIG. 8 is a plot showing that human IL-21 (hIL-2) drives human plasmacell differentiation. Purified human peripheral blood B cells werecultured at 1×10⁵ cells/100 μl with RPMI/10% FCS for 4 days. The cellswere either not stimulated (nothing; “−”) or stimulated with 10 μg/mlanti-IgM (“αIgM”), or 1 μg/ml anti-CD40(“αCD40”) in the presence orabsence of 100 U/ml hIL-2, 200 ng/ml hIL-21 or IL-2 and IL-21. At theend of the incubation, supernatants were harvested and cells werephenotypicalty characterized by flow cytometry. The cells were stainedwith IgD-PE, CD19-PercP-cy5.5 and CD38 APC. The supematants wereanalyzed for immunoglobulin content. Data represents one of severalexperiments. In addition, cells were analyzed at several days postincubation. A time dependent loss of the IgD⁺ cells following treatmentwith (a) nothing (“−”), (b) IL-21, (c) or IL-2 and IL-21, and anti-IgMwas observed; by 7 days over 80% of the cells were IgD⁻.

FIGS. 9A, C and D are scatter plots and FIG. 9B is a bar graphillustrating the ability of IL-21 to induce plasma cell differentiation.(FIG. 9A) Purified peripheral blood B Purified peripheral blood B cellswere stimulated with anti-IgM, anti-CD40, both stimuli, or neither inthe presence or absence of IL-2 and/or IL-21 as indicated. After 3 daysof culture, proliferation was determined by incubating the cells for 16hrs with ³[H]-thymidine (FIG. 9B). After 6 days of culture, the cellswere stained and analyzed for surface expression of IgD and CD38 byCD19⁺ cells (FIG. 9C). Results from a representative experiment of sixsimilar experiments are shown. (FIG. 9D) Purified B cells were firstlabeled with CFSE before being cultured. Following 7 days of culture,CD19⁺ B cells were analyzed for CD38 expression and CFSE dilution.

FIGS. 10A and B are bar graphs illustrating induction of plasma cellsare induced following co-culture with IL-21. Purified peripheral blood Bcells were cultured as described in FIG. 9C, and the mean percentage(±SEM) of plasma cells from seven independent experiments is shown (FIG.10A). The mean ³[H]-thymidine incorporation following 3 days of cultureas CPM (±SEM) for these experiments was: IL-2, 4,300 (±1,200);anti-IgM+IL-2, 7,700 (±1,800); anti-CD40+IL-2, 7,600 (±1,100); andanti-IgM and anti-CD40+IL-2, 19,700 (±10,400); IL-2+IL-21, 3,900(±1,300); anti-IgM+IL-2+IL-21, 22,300 (±3,200); anti-CD40+IL-2+IL-21,63,700 (±7,600); and anti-IgM and anti-CD40+IL-2+IL-21, 60,002 (+3,600).The absolute plasma cell number following 7 days in culture from onerepresentative experiment is also shown (B). There were approximately500 plasma cells/well in the initial population of cells.

FIGS. 11A and B are bar graphs, and FIGS. 11C and D are scatter plotsdemonstrating that IL-21 induces class switch recombination and plasmacell differentiation from both naïve and memory B cells. B cells werepositively-selected from cord blood, or negatively-selected fromperipheral blood and further purified into CD20⁺ CD27⁺ memory B cells bycell sorting. All cells were cultured with anti-IgM, anti-CD40, bothstimuli, or neither in the presence or absence of IL-2 and/or IL-21. Bcell populations were stimulated as indicated and proliferativeresponses were measured by ³[H]-thymidine incorporation after 3 days inculture of either (FIG. 11A) CD27⁻ naïve or (FIG. 11B) CD27⁺ memory Bcells. The purity of the cord blood and CD20⁺ CD27⁺ memory B cells atday 0 is shown in (FIGS. 11C and D). Neither cord blood B cells norCD20⁺ CD27⁺ memory cells contained identifiable plasma cells beforeculture. After 6 days of culture, the cells were stained and analyzedfor IgD and CD38 expression by CD19⁺ cells. No difference was observedin proliferation or change in phenotype of cord blood B cells isolatedby negative vs. positive selection. Data are representative of resultsfrom one of six experiments for purified CD27⁻ cord blood B cells andone of five similar experiments for CD27⁺ sorted peripheral blood Bcells.

FIGS. 12A-C are bar graphs illustrating that IL-21 induces secretion ofIgG and IgM. Purified B cells were cultured with either anti-IgM,anti-CD40, both stimuli, or neither in the presence or absence of IL-2,IL-21, or both IL-2 and IL-21. Cell supernatants were removed following(FIGS. 12A and B) 10-11 days of culture or (FIG. 12C) following 6 days(or 8 days, data not shown) for cord blood and 12 days for CD27⁻ & CD27⁺B cells. Production of total IgG or IgM from purified total peripheral Bcells (FIG. 12A) or cord blood B cells (FIG. 12B) and secretion ofspecific Ig isotypes from naïve cord blood B cells (CB), naïve (CD27⁻)adult B cells or memory (CD27⁺) B cells (FIG. 12C) were quantitated. In(FIGS. 12A and B), IgM levels were not measured in any culturescontaining anti-IgM. Data are mean concentration (±SD) from one of sevenand six representative experiments, for (FIGS. 12A and B) respectively,and one representative experiment of four cord blood and three withCD27⁻ and CD27⁺ adult peripheral B cells (FIG. 12C).

FIGS. 13A-C are bar graphs showing that IL-21 induces both BLIMP and AIDexpression, but not somatic hypermutation. Purified peripheral B cells(FIG. 13A) or cord blood B cells (FIGS. 13B and C) were cultured withthe stimuli and cytokines indicated. After 3 days of culture, cells wereharvested, verified for cell surface phenotype, RNA isolated andquantitative RT-PCR was carried out to identify Blimp-1, AID, Bcl-6 andPAX-5 mRNA. All data were normalized to β-2M mRNA and data shownrepresent fold change compared to samples that were incubated with IL-2with no other stimuli (FIG. 13A), or to primary cord blood B cells (FIG.13B). Data are representative of results from six similar experimentsfor peripheral blood B cells, and two for cord blood B cells. Mean foldchange (±SEM) of triplicate mRNA samples from one representativeexperiment is shown. Negatively-selected cord blood B cells wereexamined for mutations of rearranged heavy chain genes before and after7-12 days of in vitro stimulation with IL-2, IL-21 and anti-CD40 with orwithout anti-IgM (FIG. 13C). After stimulation, IgD⁻ CD38^(hi) plasmacells (n=189) or post-switched IgD⁻ CD38^(−/lo) B cells (n=79) wereisolated by cell sorting and the Ig V_(H) genes were amplified fromgenomic DNA by single cell PCR, sequenced, and the frequency of Ig V_(H)mismatches was compared to that found in cord blood B cells beforeculture (n=141). Combined results from four experiments are shown.

FIGS. 14A-C are a series of scatter plots and two bar graphs,respectively, showing that IL-4 inhibits IL-21-induced plasma celldifferentiation. Purified peripheral B cells were isolated and culturedwith the stimuli and cytokines indicated. Before culture the B cellscontained 78% naïve B cells, 20% IgD⁻ B cells and 0.4% plasma cells.Cell surface expression of IgD and CD38 by CD19⁺ B cells following 6-7days of culture is shown (FIG. 14A). IgG was quantified from culturesupernatants following 7-11 days of incubation and data are shown as amean concentration (±SEM) (FIG. 14B). mRNA was isolated and Blimp-1 andAID expression was determined following 3 days of culture (FIG. 14C).All data in (FIG. 14C) were normalized to β-2M mRNA and shown as foldchange compared to cultures incubated with only IL-2 and IL-4. Data areshown as a mean fold change (±SEM). Data are representative of resultsfrom cultures of (FIG. 14A) two similar experiments with SAC, three withαIgM, five with αCD40, seven with αIgM & αCD40, (FIG. 14B) two with SAC,and eight with αIgM and αCD40. For anti-CD40 stimulation, three of sixexperiments are shown in which four resulted in IL-4-induced suppressionof IgG production and two did not which was not time dependent (FIG.14B). The data in FIG. 14C are representative of four similarexperiments.

FIGS. 15A-C are a series of scatter plots and two bar graphs,respectively, showing that IL-10 does not synergize with IL-21 in thedifferentiation of plasma cells and IgG production. Purified naïve cordblood B cells or CD27⁺ memory peripheral blood B cells were culturedwith the stimuli and cytokines indicated for 6 days for cord blood Bcells and 7 days for CD27⁺ peripheral blood B cells. At the end of theincubation period, cells were stained and analyzed for IgD and CD38expression by CD19⁺ cells (FIG. 15A). Data are representative of resultsfrom one of three similar experiments. Cell supernatants were removed atend of culture and total IgG determined by ELISA (FIG. 15B). Data areshown as a mean concentration (±SD). Neither cord blood nor CD20⁺CD27⁺memory B cells contained plasma cells before culture.

FIGS. 16A-D are bar graphs illustrating proliferative responses andimmunoglobulin secretion induced by IL-21 or IL-2 plus IL-10. IL-21induces greater proliferation, plasma cell differentiation, and IgGproduction from anti-CD40-stimulated peripheral blood and cord blood Bcells than does IL-2 and IL-10. (A-C) Purified naïve cord blood B cells(CB) or (A-D) peripheral blood B cells (PB) were cultured with thestimuli and cytokines indicated for (A) 3 days, (B) 7 days, (C) 9 daysfor CB B cells and 10 days for PB B cells and (D) 3 days. At the end ofthe incubation period (A) ³[H]-thymidine incorporation, (B) absoluteplasma cell number, and (C) IgG secretion were determined. In (D)relative fold change of mRNA was determined for BLIMP-1 and AID. Allsamples were first normalized to β-2M mRNA and then to mRNA of samplesthat were not stimulated and did not contain cytokine (nil, nil). Dataare shown as mean fold change (±SEM). Data shown are representativeresults.

FIG. 17 is a set of histograms illustrating the cell surface phenotypeof IL-21-induced plasma cells. Purified peripheral blood B cells wereisolated and cultured with IL-21, anti-IgM and anti-CD40. Following 7 or8 days of culture, CD19⁺ cells were subdivided into IgD—CD38^(lo) cells(dark grey histogram) and IgD-CD38^(hi) plasma cells (light greyhistogram) and further analyzed for expression of the markers indicated.The data reflect representative results.

FIGS. 18A, B and C are histograms showing that the majority ofIL-21-induced plasma cells are not in cell cycle. Purified peripheralblood B cells were isolated and cultured with IL-21, anti-IgM andanti-CD40. Following 7 days of culture, cells were stained with thecombination of either anti-IgD, anti-CD27, anti-CD19 and anti-CD38 oranti-CD27, anti-CD38 and PI. For the latter, cells were subdivided intoeither CD27− & +, CD38^(lo) non-plasma cells (non-PC) or CD27^(hi),CD38^(hi) plasma cells (PC) and cell cycle was determined. Cell surfacephenotype of cells before culture is shown.

FIGS. 19A-C are a bar graph, scatter plots and line graph, respectivelyillustrating that Ig production from IL-21-induced plasma cells isresistant to hydroxyurea. Purified peripheral blood B cells wereisolated and (A) cultured in the presence of IL-21 with or withoutanti-CD40 and anti-IgM and with or without hydroxyurea (HU, 1×10⁻²M) asindicated. Following 3 days of culture, proliferation was determined byincubating the cells for 16 hrs with ³[H]-thymidine. (B) Purifiedperipheral blood B cells were isolated and cultured with or withoutIL-21 in the presence of anti-CD40 and anti-IgM and hydroxyurea wasadded (or not) on the days indicated. The cultures were analyzed for IgDand CD38 expression by CD19⁺ B cells after 8 or 11 days in culture asindicated. (C) Supernatants from IL-21, anti-IgM andanti-CD40-stimulated cultures in (B) were analyzed for IgG content. HUwas added where indicated. Data are representative of results frommultiple experiments.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

-   -   SEQ ID NO: 1 is the amino acid sequence of human IL-21.    -   SEQ ID NO: 2 is the amino acid sequence of murine IL-21.    -   SEQ ID NOs: 4-16 are the nucleic acid sequences of        oligonucleotides used in the experimental studies described in        the Examples.

DETAILED DESCRIPTION

Within the B cell lineage, IL-21 is important for IgG1 production andcooperates with IL4 for the production of other antibody classes in vivo(Ozaki et al., Science 298:1630-1634, 2002; Suto et al., Blood100:4565-4573). IL-21R^(−/−) mice have markedly diminished IgG1 butgreatly elevated IgE levels in response to antigen, andIL-21R^(−/−)IL-4^(−/−) double knockout mice have a severely impaired IgGand IgE response. In vitro, IL-21 can enhance the proliferative responseof human and murine B cells stimulated with antibodies to CD40, but itinhibits B cell proliferation in response to anti-IgM+IL4 or LPS(Parrish-Novak et al., Nature 408:57-63, 2000), and correspondingly canaugment B cell death (Mehta et al. J. Immunol. 170:4111, 2003; see alsoU.S. Provisional Patent Application No. 60/393,215; U.S. ProvisionalPatent Application No. 60/449,056; PCT Application No. PCT/US 03/20370,all of which are incorporated by reference in their entirety).

As disclosed herein, whereas IL-21 induces death of resting mature Bcells, it promotes the differentiation of B cells activated in a T celldependent manner into memory and plasma cells. IL-21 exerts thesedifferential effects on B cell fate depending on the signaling context.Thus, IL-21 serves as a master regulator of B cell maturation andterminal differentiation into memory B cells and plasma cells.

IL-21 has been identified in a number of species, and any of these canbe employed in the methods described herein. Preferably, an IL-21 isselected that binds to an IL-21 receptor expressed on the target B cellprogenitor or mature B cell. Similarly, IL-21 polypeptide variants,especially conservative variants having only a small number of, such as1 or 2 or 3, amino acid substitutions, relative to a naturally occurringIL-21 can be employed in the methods described herein. Additionally,IL-21 analogs, regardless of their amino acid composition, includingsubsequences and fragments of IL-21 are favorably employed in themethods described herein, provided that the IL-21 analog retains atleast partial functional activity of an IL-21. That is, analogs that areIL-21 agonists, which bind to the IL-21 receptor and produce aphysiological effect produced by binding of a native IL-21 to itscognate receptor, can be utilized in the same manner as an IL-21polypeptide in the methods described herein.

The present disclosure provides methods for inducing differentiation ofmemory B cells and plasma cells from B cell progenitors and mature Bcells, in quantities suitable for isolation of memory B cells and plasmacells. A population of cells including B cell progenitors and/or mature,antigen specific IgD expressing, B cells are contacted with acomposition including IL-21. Following exposure to an effective amountof IL-21, B cell progenitors and mature B cells preferentiallydifferentiate into memory B cells and plasma cells. The resultingenrichment for memory B cells and plasma cells permits isolation these Bcell classes with increased efficiency.

For example, a population of bone marrow derived cells or peripheralblood cells including a wide variety of cell types including B cellprogenitors and mature B cells can be exposed to an IL-21 containingcomposition, thereby inducing differentiation of B cell progenitors andmature B cells into memory B cells and/or plasma cells. Optionally, Bcell progenitors and/or mature B cells can be isolated prior to treatingthem with IL-21. The methods described herein are applicable to B cellsderived from a variety of species, particularly mammals, includinghumans.

Also described are methods for inducing differentiation of B cellprogenitors into memory B cells and plasma cells by contacting apopulation of progenitor cells with an agent that activates at least onemember of the JAK/STAT signaling pathway, for example JAK1, JAK3, STAT5Aand/or STAT5B. Activation of this signaling pathway inducesdifferentiation of memory B cells and plasma cell, which can be isolatedfrom the population of treated cells. IL-21 is an exemplary agent thatactivates the JAK/STAT signaling pathway, thereby inducingdifferentiation of B cells into memory B cells and plasma cells.

As disclosed herein, IL-21 preferentially promotes differentiation of Bcells into mature subsets with desirable functional attributes. Thus,IL-21 is of use to enhance an immune response in a subject, including ahuman subject. Although these methods have widespread applicability toenhance the efficacy of an immune response in a subject, these methodscan also be employed more particularly to ameliorate immunodeficiencies,especially a deficiency characterized by reduction in number or functionof memory B cells and/or plasma cells. For example, the methodsdescribed herein for enhancing an immune response can be used to treat asubject with a post-transplantation B cell deficiency.

An immune response in a subject can be enhanced by contacting apopulation of cells including mature B cells and/or B cell progenitorswith a composition containing IL-21 to induce differentiation of themature B cells or B cell progenitors into memory B cells and/or plasmacells. The differentiated mature B cells and plasma cells are thenisolated and introduced into a subject to enhance an immune response.

For example, the cells can be contacted with IL-21 by administering acomposition containing IL-21 directly to the subject, such as a humansubject. In this case, the IL-21 is administered in a pharmaceuticallyacceptable formulation, such as a formulation containing IL-21 and apharmaceutically acceptable carrier or excipient. Alternatively, thecells can be contacted with IL-21 ex vivo.

In some cases, the population of cells including mature B cells and/or Bcell progenitors, such as immature B cells, is isolated. For example,mature B cells and/or B cell progenitors can be isolated from peripheralblood or bone marrow.

Optionally, the cells can also be contacted with an antigen, such as anantigen derived from a pathogen (e.g., a bacterial antigen, a viralantigen, or an antigen from a parasite).

Thus, the present disclosure provides methods for treating a subjectwith a condition characterized by a specific deficiency of memory Bcells and/or plasma cells by administering to the subject atherapeutically effective amount of IL-21 or an agonist thereof, therebyameliorating a sign or symptom of the deficiency. For example, themethods described herein can be used to ameliorate the symptoms of animmunodeficiency, such as an acquired immunodeficiency, e.g., a postbone marrow transplantation deficiency. Such an immunodeficiency can becharacterized by a reduction in the number and/or function of memory Bcells and plasma cells. Administering a therapeutically effective amountof IL-21 increases the number and/or the proportion of at least one ofmemory B cells and plasma cells.

In an embodiment, the IL-21 is administered by treating a population ofcells including one or more of mature B cells and B cell progenitors exvivo. Optionally, the memory B cells and/or plasma cells induced todifferentiate ex vivo are isolated and introduced into the subject. Thetreated population of cells can be returned to the same subject, or canbe introduced into a different subject, that is, the cells can beintroduced as an autologous transfer or as a heterologous transfer.

Based on the identification of IL-21 as an agent that preferentiallyinduces differentiation of memory B cells and plasma cells, methods havebeen developed for identifying agents that exert a physiological effecton the differentiation of these mature B cell subsets from B cellprogenitors. Accordingly, methods for identifying agents with aphysiological effect on the differentiation of memory B cells and plasmacells are described herein. Such methods involve contacting an isolatedpopulation of cells (including B cell progenitors, such as immature Bcells) that has been exposed to IL-21 with an agent, and detecting aphysiological effect of the agent on memory B cell differentiation,plasma cell differentiation, or both. For example, the effect detectedcan be inhibition of differentiation of one or more of memory B cellsand plasma cells. Typically, screening methods for identifying agentswith a physiological effect on differentiation of memory B cells andplasma cells involve contacting each of a plurality of populations ofcells (or subsets of a population of cells) with a different agent. Thedifferent agents are usually members of a library of compositions.

Also disclosed are methods for identifying an agent that inhibits anactivity of IL-21. The methods involve contacting a cell with at leastone agent and detecting a decrease in the production or activity of atleast one of Blimp-1, AID and Bcl-6 relative to a control cell.Optionally, the cell is contacted with IL-21. In one example, the methodis utilized to identify antibodies that specifically bind to Blimp-1,AID or Bcl-6. Commonly, the control cell is a cell that is not contactedwith the agent.

These and other features of the disclosure will be apparent upon reviewof the following detailed description and examples.

I. Abbreviations

-   -   β-2M: β-2 microglobulin    -   γ_(C): common cytokine receptor gamma (γ) chain    -   Ab: antibody    -   AID: activation induced cytidine deaminase    -   Bcl-6: B cell lymphoma 6    -   BCR: B-cell receptor    -   Blimp-1: B-lymphocyte induced maturation protein 1    -   CD40L: CD40 ligand    -   CSR: class switch recombination    -   FACS: fluorescence activated cell sorting or scanning    -   Hr: hour    -   IL-4: interleukin-4    -   IL-21: interleukin-21    -   IL-21R: interleukin 21 receptor    -   Ig: immunoglobulin    -   JAK: Janus Activated Kinase    -   LPS: lippopolysaccharide    -   FO: lymphoid follicles    -   FZ: follicular zone    -   MZ: marginal zone    -   M: mature    -   NF: newly formed    -   PARP: poly (ADP ribose) polymerase    -   SAC: Staphylococcus aureus Cowan I    -   SCID: severe combined immunodeficiency disease    -   S.D.: standard deviation    -   SH2: SRC homology-2    -   SMH: somatic hypermutation    -   STAT: Signal Transducer and Activator of Transcription    -   TG: transgenic    -   TUNEL: TdT-dependent dUTP-biotin nick end labeling    -   μg: microgram    -   WT: wild-type        II. Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. For example,definitions of common terms in molecular biology can be found inBenjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. The term“comprises” means “includes.” For example, “comprising A or B” meansincluding A or B, or both A and B, unless clearly indicated otherwise.The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example.”

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Agent: Any polypeptide, compound, small molecule, organic compound,salt, polynucleotide, or other molecule of interest.

Agonist: An agent that has affinity for and stimulates physiologicactivity at cell receptors normally stimulated by naturally occurringsubstances, thus triggering a biochemical response. An IL-21 and/orIL-21 receptor agonist has affinity for the IL-21 receptors andstimulates an activity induced by the binding of IL-21 with itsreceptor. In contrast, an “antagonist” is an agent that has affinity forand blocks or inhibits activity of a cell receptor normally stimulatedby a naturally occurring substance. Accordingly, an IL-21/IL-21 receptorantagonist binds to the IL-21 receptor and inhibits an activity normallyinduced by IL-21.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals, primates, and birds.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen. Inone embodiment the antigen is the IL-4 or IL-21. In another embodiment,the antigen is the IL-4 or the IL-21 receptor.

A naturally occurring antibody (e.g., IgG) includes four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. However, the antigen-binding function of an antibodycan be performed by fragments of a naturally occurring antibody. Thus,these antigen-binding fragments are also intended to be designated bythe term antibody. Examples of binding fragments encompassed within theterm antibody include (i) an Fab fragment consisting of the VL, VH, CLand CH1 domains; (ii) an Fd fragment consisting of the VH and CH1domains; (iii) an Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (iv) a dAb fragment (Ward et al., Nature341:544-6, 1989) which consists of a VH domain; (v) an isolatedcomplimentarity determining region (CDR); and (vi) an F(ab′)₂ fragment,a bivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region. Furthermore, although the two domains of theFv fragment are coded for by separate genes, a synthetic linker can bemade that enables them to be made as a single protein chain (known assingle chain Fv (scFv); Bird et al. Science 242:423-6, 1988; and Hustonet al., Proc. Natl. Acad. Sci. USA 85:5879-83, 1988) by recombinantmethods. Such single chain antibodies are also included.

Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described for wholeantibodies. An antibody is further intended to include bispecific andchimeric molecules that specifically bind the target antigen. Antibodiescan be derived from any species, such as fully human antibody.Antibodies can be chimeric, such as a humanized mouse monoclonalantibody.

“Specifically binds” refers to the ability of individual antibodies tospecifically immunoreact with an antigen, such as I1-4, I1-21, the IL-4receptor, or the IL-21 receptor, or Bcl-6 or Blimp-1. The binding is anon-random binding reaction between an antibody molecule and anantigenic determinant of the target cytokine or receptor. The desiredbinding specificity is typically determined from the reference point ofthe ability of the antibody to differentially bind the target cytokineor receptor and an unrelated antigen, and therefore distinguish betweentwo different antigens, particularly where the two antigens have uniqueepitopes. An antibody that specifically binds to a particular epitope isreferred to as a “specific antibody”.

B Cell: A subset of lymphocytes, that is, white blood cells(leukocytes). Mature B cells differentiate into plasma cells, whichproduces antibodies, and memory B cells. A “B cell progenitor” is a cellthat can develop into a mature B cell. B cell progenitors include stemcells, early pro-B cells, late pro-B cells, large pre-B cells, smallpre-B cells, and immature B cells and transitional B cells. Generally,early pro-B cells (that express, for example, CD43 or B220) undergoimmunoglobulin heavy chain rearrangement to become late pro B and pre Bcells, and further undergo immunoglobulin light chain rearrangement tobecome an immature B cells. Immature B cells include T1 and T2 B cells.For example, in mice, immature B cells include T1 B cells that areAA41^(hi)CD23^(lo) cells. Another example of a mouse immature B cell isa T2 B that is an AA41^(hi)CD23^(hi) cell. In humans, immature B cells(for example, immature peripheral transitional B cells) includeCD38^(hi), IgD⁺, CD10⁺, CD24^(hi), CD44^(lo), CD23^(lo) and CD1^(lo)cells. Thus, immature B cells include B220 (CD45R) expressing cellswherein the light and the heavy chain immunoglobulin genes arerearranged. In one embodiment, immature B cells express CD45R, class II,IgM, CD19 and CD40. Immature B cells do not exhibit surrogate lightchain expression, but do express Ig αβ and RAG. Immature B cells candevelop into mature B cells, which can produce immunoglobulins (e.g.,IgA, IgG or IgM). Mature B cells have acquired surface IgM and IgD, arecapable of responding to antigen, and express characteristic markerssuch as CD21 and CD23 (CD23^(hi)CD21^(hi) cells). B cells can beactivated by agents such as lippopolysaccharide (LPS) or IL-4 andantibodies to IgM. Common biological sources of B cells and B cellprogenitors include bone marrow, peripheral blood, spleen and lymphnodes. Plasma cells are terminally differentiated B cells that are thepredominant antibody-secreting cells. Memory B cells are small;long-lived B lymphocytes produced following antigen stimulation.Typically, memory B cells express high affinity antigen specificimmunoglobulin (B cell receptor) on their cell surface.

Bcl-6: A sequence-specific DNA binding transcriptional repressor. Bcl-6is a 706-amino-acid nuclear zinc finger protein. Bcl-6 is implicated inthe formation of germinal centers and Th2 mediated responses. The Bcl-6locus is the breakpoint cluster region in B. cell lymphomas. The Bcl-6locus is of 30 kilobases in length containing at least a Bcl-6 genewhich codes for a protein. Therefore, the Bcl-6 locus contains both the5′ and 3′ flanking region of the coding sequences of the Bcl-6 gene.Antibodies to this protein stain the germinal center cells in lymphoidfollicles, the follicular cells and interfollicular cells in follicularlymphoma, some extrafollicular T cells, diffuse large B cell lymphomas,and Burkitt's lymphoma, and the majority of the Reed-Sternberg cells innodular lymphocyte predominant Hodgkin's disease. Bcl-6 expression isseen in approximately 45% of CD30+ anaplastic large cell lymphomas butis absent in other peripheral T cell lymphomas. The amino acid sequenceof Bcl-6 is available on the internet, for example it can be found asEMBL Accession No. Z21943, GENBANK® Accession No. U00115, or asSwissProt database as Accession No. P41182.

Blimp-1: A protein that acts as a repressor of beta-interferon geneexpression. The protein binds specifically to the PRDI (positiveregulatory domain I element) of the beta-IFN gene promoter.Transcription of this gene increases upon virus induction. Twoalternatively spliced transcript variants that encode different isoformshave been reported. The sequence of Blimp-1 can be found on theinternet, for example it can be found as GENBANK® Accession No.NP_(—)001189.

CD45: An antigen also known as Leukocyte common antigen or T200. CD45 isa tyrosine phosphatase that augments signaling through antigen receptorof B and T cells. There are multiple isoforms of CD45 (such as CD45RO,CD45RA, CD45RB, CD45RC and CD45RO) that result from alternativesplicing. CD45RO Lacks the A, B, and C exons of CD45, while CD45RA,CD45RB, and CD45RC contain the A, B and C exons respectively. CD45RO,CD45RA, and CD45RB are expressed on B cells. Expression of CD45RC isrestricted to T cells. Mouse B cells are characterized by expression ofan isoform of CD45 designated CD45R or B220, uniquely detected by themonoclonal antibody 6B2.

Costimulation: An event or stimulus in addition to antigen bindinginvolved in proliferation or activation of lymphocytes. A costimulationagent is any agent that is capable of promoting proliferation oractivation of a lymphocyte. Costimulation agents include lymphocytespecific molecules, such as molecules expressed on the lymphocyte cellsurface or soluble forms, or functional agonists thereof, that incombination with binding of antigen or another ligand (such as anantibody that specifically binds to an immunoglobulin) to a cell surfaceantigen receptor, are capable of inducing proliferation of a lymphocyte.Costimulation agents can also include non-specific lymphocyte activatingagents, such as LPS and Staphylococcus aureus Cowan I antigen (SAC).Exemplary B cell costimulation agents include agents that bind to CD40on the surface of a B cell, such as CD40 ligand or anti-CD40 antibody.Exemplary T cell costimulation agents include agents that bind to CD28and/or CTLA-4, such as B7 and B7.2.

In certain examples disclosed herein, an antibody that specificallybinds to CD40 expressed on the surface of a B cell is used as acostimulation agent. In other examples, a polyclonal activators, such asLPS or SAC is used as a costimulation agent (for example, in the absenceof antigen). Thus, in certain examples, a B cell progenitor (such as animmature B cell or a transitional B cell), a naïve mature B cell, amemory cell, or a population of cells including at least one of thesecell types, is contacted with IL-21 in the presence of a costimulationagent. The costimulation agent can be administered from an exogenoussource or can be provided by one or more cells in a mixed population ofcells, either in vitro or in vivo.

Cytokine/Interleukin (IL): A generic name for a diverse group of solubleproteins and peptides which act as humoral regulators at nano- topicomolar concentrations and which, either under normal or pathologicalconditions, modulate the functional activities of individual cells andtissues. These proteins also mediate interactions between cells directlyand regulate processes taking place in the extracellular environment.Many growth factors and cytokines act as cellular survival factors bypreventing programmed cell death. Cytokines and interleukins includeboth naturally occurring peptides and variants that retain full orpartial biological activity. Although specific cytokines/interleukinsare described in the specification, they are not limited to thespecifically disclosed peptides.

Deletion: The removal of a sequence of nucleic acid, such as DNA.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid, RNA). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine, and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides,referred to as codons, in DNA molecules code for amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequences of three nucleotides in the mRNA into which theDNA sequence is transcribed.

Electroporation: A method of inducing or allowing a cell to take upmacromolecules by applying electric fields to reversibly permeabilizethe cell walls. Various methods and apparatuses used are further definedand described in: U.S. Pat. No. 4,695,547; U.S. Pat. No. 4,764,473; U.S.Pat. No. 4,882,281; U.S. Pat. No. 4,946,793; U.S. Pat. No. 4,906,576;U.S. Pat. No. 4,923,814; and U.S. Pat. No. 4,849,089.

Eukaryotic cell: A cell having an organized nucleus bounded by a nuclearmembrane. These include simpler organisms such as yeasts, slime molds,and the like, as well as cells from multicellular organisms such asinvertebrates, vertebrates, and mammals. A eukaryotic cell can be, forexample, an endothelial cell, a smooth muscle cell, an epithelial cell,a hepatocyte, a cell of neural crest origin, a tumor cell, ahematopoetic cell, an immunologic cell, (e.g., a T cell, a B cell, amonocyte, a macrophage, a dendritic cell), a fibroblast, a keratinocyte,a neuronal cell, a glial cell, an adipocyte, a myoblast, a myocyte, achondroblast, a chondrocyte, an osteoblast, an osteocyte, an osteoclast,a secretory cell, an endocrine cell, an oocyte, and a spermatocyte.These cell types are described in standard histology texts, such asMcCormack, Introduction to Histology,© 1984 by J.P. Lippincott Co.;Wheater et al., eds., Functional Histology, 2nd Ed.,© 1987 by ChurchillLivingstone; Fawcett et al., eds., Bloom and Fawcett: A Textbook ofHistology,© 1984 by William and Wilkins.

Gene: A functional DNA sequence. For example, a gene can include controland/or coding sequences necessary for the production of an RNA or apolypeptide, such as a protein. Where a gene encodes a polypeptide, thepolypeptide can be encoded by a full-length coding sequence or by anyportion (or subsequence) of a full length coding sequence, so long as atleast a part of the functional activity of the polypeptide is retained.

Heterologous or foreign gene: A gene that is introduced into the genomeof a cell or organism, such as a multicellular animal, by experimentalmanipulations and can include polynucleotide sequences found in thatcell or organism so long as the introduced gene contains somemodification (e.g., a point mutation, the presence of a selectablemarker gene, a non-native regulatory sequence, or a native sequenceintegrated into the genome at a non-native location, etc.) relative tothe naturally-occurring gene. More broadly, a heterologous or foreignnucleic acid is any nucleic acid regardless of its functional attributesthat does not originate in the cell or organism in which it is located,or does not exist in association with the nucleic acids in which it isassociated upon introduction into the cell or organism.

Interleukin (IL)₄: IL-4 is a protein produced mainly by a subpopulationof activated T cells (CD4⁺TH2 cells), which also secrete IL-5 and IL-6.IL-4 is 129 amino acids (20 kDa) that is synthesized as a precursorcontaining a hydrophobic secretory signal sequence of 24 amino acids.IL-4 is glycosylated at two arginine residues (positions 38 and 105) andcontains six cysteine residues involved in disulfide bond formation.Some glycosylation variants of IL-4 have been described that differ intheir biological activities. A comparison of murine and human IL-4 showsthat both proteins only diverge at positions 91-128.

The human IL-4 gene contains four exons and has a length ofapproximately 10 kb. It maps to chromosome 5q23-31, while the murinegene maps to chromosome 11. At the nucleotide level the human and themurine IL-4 gene display approximately 70% homology.

The biological activities of IL-4 are species-specific; mouse IL-4 isinactive on human cells and human IL-4 is inactive on murine cells. IL-4promotes the proliferation and differentiation of activated B cells, theexpression of class II MHC antigens, and of low affinity IgE receptorsin resting B cells. In addition, IL-4 is known to enhance expression ofclass II MHC antigens on B cells. This cytokine also can promote the Bcells' capacity to respond to other B cell stimuli and to presentantigens for T cells.

The classical detection method for IL-4 is a B cell costimulation assaymeasuring the enhanced proliferation of stimulated purified B cells.IL-4 can be detected also in bioassays, employing IL4-responsive cells(e.g., BALM4, BCL1, CCL-185, CT.4S, amongst others). A specificdetection method for human IL-4 is the induction of CD3 in a number of Bcell lines with CD23 detected either by flow-through cytometry or by afluorescence immunoassay. An alternative and entirely differentdetection method is RT-PCR (for review see: Boulay and Paul, CurrentOpinion in Immunology 4:294-8, 1992; Paul and Ohara, Annual Review ofImmunology 5:429-59, 1987).

IL-21: A cytokine cloned from a cDNA library derived from activated CD3+T cells (Parrish-Novak et al., Nature 408:57-63, 2000). The IL-21 cDNAencodes a secreted protein of 131 amino acids protein most closelyrelated to IL-2 and IL-15. The IL-21 gene has been mapped to humanchromosome 4q26-q27 near the IL-2 gene.

IL-21 mRNA has been demonstrated to be expressed in activated CD4+cells, but not in other T cells, B cells, or monocytes (Parrish-Novak etal., Nature 408:57-63, 2000). However, it has been demonstrated thatIL-21 stimulates proliferation of B cells that are stimulated bycross-linking of the CD40 antigen and proliferation of B cellsstimulated by IL-4 in addition to anti-IgM. IL-21 has also been shown tostimulate proliferation of naive (CD45RA (+)) cells, mediated byengagement of CD3. IL-21 has also been shown to stimulate theproliferation of bone marrow progenitor to cells and to enhance theexpression of the NK cell marker CD56 in the presence of IL-15. (Forreview, see Horst Ibelgaufts' COPE: Cytokines Online PathfinderEncyclopedia, available on the internet).

The IL-21 receptor has been isolated and was found to be expressed byCD23+ B cells, B cell lines, a T cell leukemia line, and NK cell lines.The receptor gene has been mapped to human chromosome 16p12 (seeParrish-Novak et al., Nature 408:57-63, 2000; Ozaki et al., Proc. Natl.Acad. Sci. USA 97:11439-11444, 2000).

The receptor, which is 538 amino acids in length, is most closelyrelated to human IL-2 beta receptor, and contains a WSXWS motif in theextracellular region, typical of type-1 cytokine receptors.

Immunoglobulins: A class of proteins found in plasma and other bodyfluids that exhibits antibody activity and binds with other moleculeswith a high degree of specificity; divided into five classes (IgM, IgG,IgA, IgD, and IgE) on the basis of structure and biological activity.The IgG class has been further divided into the IgG1, IgG2a, IgG2b, IgG4and IgG4 subtypes. Immunoglobulins and certain variants thereof areknown and many have been prepared in recombinant cell culture (e.g., seeU.S. Pat. No. 4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP256,654; EP 120,694; EP 125,023; Faoulkner et al., Nature 298:286, 1982;Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol2:239, 1984).

A native (naturally occurring) immunoglobulin is each is made up of fourpolypeptide chains. There are two long chains, called the “heavy” or “H”chains which weigh between 50 and 75 kilodaltons and two short chainscalled “light” or “L” chains weighing in at 25 kilodaltons. They arelinked together by what are called disulfide bonds to form a “Y” shapemolecule. Each heavy chain and light chain can be divided into avariable region and a constant region. An Fc region includes theconstant regions of the heavy and the light chains, but not the variableregions.

Isolated or purified: An isolated biological component (such as anucleic acid, peptide or protein disclosed herein) has beensubstantially separated, produced apart from, or purified away fromother biological components in the cell of the organism in which thecomponent naturally occurs, such as other chromosomal andextrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides andproteins that have been isolated include nucleic acids and proteinspurified by standard purification methods. The term also embracesnucleic acids, peptides, and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids. Isolatedor purified compositions can be produced, for example, by standardpurification techniques, or by recombinant techniques. In someembodiments, a preparation of a polypeptide is purified such that theprotein represents at least 50%, for example at least 70%, of the totalpolypeptide content of the preparation.

An “isolated” cell is a cell that has been purified from the othercellular components of a tissue. Cells can be isolated by mechanicaland/or enzymatic methods. In several embodiments, an isolated populationof cells includes greater than about 80%, about 85%, about 90%, about95%, or greater than about 99% of the cells of interest. In anotherembodiment, an isolated population of cells is one in which no othercells of a different phenotype can be detected. In a further embodiment,an isolated population of cells is a population of cells that includesless than about 20%, about 15%, about 10%, about 5%, or less than about1% of a cells of a different phenotype than the cells of interest.

Janus Activated kinase (JAK)/Signal Transducer and Activator ofTranscription (STAT): JAKs are cytoplasmic tyrosine kinases that areeither constitutively associated with cytokine receptors or recruited toreceptors after ligand binding. In either case, stimulation with theligand results in the catalytic activiation of receptor-associated JAKs.This activation results in the tyrosine phosphorylation of cellularsubstrates, including the JAK-associated cytokine receptor chains. Someof these phosphoylated tyrosines can serve as coding sites for STATproteins, which bind to the phsphotyrosines by their SRC-homology 2(SH2) domains. STAT proteins are also phosphylated on a conservedtyrosine residue, resulting in their dimerization and acquisition ofhigh-affinity DNA-binding activity, which facilitates their action asnuclear transcription factors.

The JAK/STAT pathway is one of the most rapid cytoplasmic to nuclearsignaling mechanisms. There are a total of four JAK (JAK1-3 and tyrosinekinase 2) and seven STAT proteins (STAT1-4, STAT5A, STAT5b and STAT6).JAKs are relatively large cytoplasmic kinases of about 1,100 amino acidsin length, and range in size from about 116 kDa to about 140 kDa.Binding of IL-21 activates a JAK/STAT signaling pathway. SpecificallyIL-21 activates JAK1 and JAK3, which then phosphorylate cellularsubstrates, including one of the IL-21 receptor chains. This allowsrecruitment of STAT 5A and STAT5B proteins to the phosphorylatedreceptor by their SH2 domains, which in turn, are also phosphorylated.The STAT proteins can dimerize, translocate to the nucleus, and bindDNA. Binding of the STAT proteins to the DNA results in transcriptionbeing activated (for review see Leonard, Nature Reviews 1: 200-208,2001).

Lineage specific marker: A marker that is expressed by a specificpopulation of cells. In one embodiment, the cells are cells of a bloodvessel, other than endothelial cells, such as smooth muscle cells. Inanother embodiment, the cells are a population of immune cells, such aslymphocytes. In one specific, non-limiting example, the marker is a Bcell specific marker, such as B220. In another specific, non-limitingexample, the marker is a T cell specific marker, such as CD3, CD4, orCD8.

Mammal: This term includes both human and non-human mammals. Similarly,the terms “subject,” “patient,” and “individual” include human andveterinary subjects.

Memory and Plasma B Cells: After a B cell progenitor (e.g., apre-committed small lymphocyte) is stimulated by an antigen, itdifferentiates into a blast cell, which differentiates into an immatureplasma cell that can differentiate into either a mature plasma cell or amemory B cell. A mature plasma cell secretes immunoglobulins in responseto a specific antigen.

A memory B cell is a B cell that undergoes isotype switching and somatichypermutation that are generally found during a secondary immuneresponse (a subsequent antigen exposure following a primary exposure)but can also be detected during a primary antigen response. Generationof memory B cells generally requires helper T cells. The development ofmemory B cells takes place in germinal centers (GC) of lymphoidfollicles where antigen-driven lymphocytes undergo somatic hypermutationand affinity selection, presumably under the influence of helper Tcells.

Neutralizing amount: An amount of an agent sufficient to decrease theactivity or amount of a substance to a specified level, for example, toan undetectable level, using a standard method.

Nucleic acid: A biological polymer, a polynucleotide, consisting ofdeoxyribonucleotides and/or ribonucleotides, for example, a DNA or anRNA polymer. The term nucleic acid also includes polymers comprisingboth deoxyribonucleotides and ribonucleotides, and/or syntheticanalogues thereof.

Oligonucleotide: A linear polynucleotide sequence of up to about 200nucleotides in length, for example a polynucleotide (such as DNA or RNA)which is at least 6 nucleotides, for example at least 12, 15, 20, 30,50, 100 or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked to asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a coding sequence is operably linked to apromoter if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary, for example, to join two protein coding regions,in the same reading frame.

Pharmaceutically acceptable carriers (excipients): The pharmaceuticallyacceptable carriers useful with the methods described herein areconventional. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 15th Edition (1975), describes compositionsand formulations suitable for pharmaceutical delivery of the cytokinesand cells disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polynucleotide: A biological polymer in which the monomers arenucleotides, such as ribonucleotides, deoxyribonucleotides or acombination thereof. Optionally, a polynucleotide can include one ormore nucleotide analog. The term polynucleotide encompasses a nucleotidepolymer of any length. Therefore, a polynucleotide includes moleculesthat are at least 15, 25, 50, 100, or 200 (oligonucleotides) and alsonucleotides as long as a full-length cDNA, e.g., several kilobases orlonger. The term polynucleotide sequence refers to the sequential arrayof nucleotides in a polynucleotide. Typically, a polynucleotide sequenceis represented as a series of letters (a, c, g, and t or u) each ofwhich represents a deoxyribonucleotide or a ribonucleotide (that is,adenine, cytosine, guanine, thymine and uracil, respectively).

Polypeptide: A biological polymer in which the monomers are amino acidresidues that are joined together through amide bonds. When the aminoacids are alpha-amino acids, either the L-optical isomer or theD-optical isomer can be used, the L-isomers being preferred in nature.The term polypeptide or protein as used herein encompasses any aminoacid sequence and includes, but is not limited to, modified sequencessuch as ADP-ribosylated proteins, ribosyl-proteins, and glycoproteins.The term polypeptide is specifically intended to cover naturallyoccurring proteins, such as IL-21, as well as polypeptides (e.g., IL-21)that are recombinantly or synthetically produced.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Any cDNA sequence variant will preferably introduce no more than twenty,and preferably fewer than ten amino acid substitutions into the encodedpolypeptide. For example, an IL-21 variant polypeptide can have as manyas ten or five amino acid substitutions, but often will have no morethan three, or two or one amino acid substitutions. In some instances,an IL-21 polypeptide will have 1, or 2, or 3, or more, conservativeamino acid substitutions. Variant amino acid sequences can, for example,be 80%, 90% or even 95% or 98% identical to the native amino acidsequence. Programs and algorithms for determining percentage identitycan be found at the NCBI website.

Sample (Biological sample): Includes biological samples containingfluids, tissues, cells, and subcomponents thereof, such as DNA, RNA, andproteins. For example, common samples in the context of the presentinvention include bone marrow, spleen, lymph node, blood, e.g.,peripheral blood (but can also include any other source from which Bcells or B cell progenitors can be isolated, including: urine, saliva,tissue biopsy, surgical specimens, fine needle aspirates, autopsymaterial, and the like).

Sequence identity: For any sequences disclosed herein, the similaritybetween two nucleic acid sequences, or two amino acid sequences, isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologues of an IL-2 receptor polypeptide or a gene encoding an IL-21receptor polypeptide, will possess a relatively high degree of sequenceidentity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp,CABIOS 5:151, 1989; Corpet, et al., Nucleic Acids Research 16:10881,1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.Altschul et al., Nature Genet., 6:119, 1994, presents a detailedconsideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Specific binding agent: An agent that binds substantially only to adefined target. Thus an IL-21 receptor specific binding agent is anagent that binds substantially to an IL-21 receptor. In one embodiment,the specific binding agent is a monoclonal or polyclonal antibody thatspecifically binds the IL-21 receptor.

The term “specifically binds” refers with respect to an antigen, such asthe IL-21 receptor, to the preferential association of an antibody orother ligand, in whole or part, with a cell or tissue bearing thatantigen and not to cells or tissues lacking that antigen. It is, ofcourse, recognized that a certain degree of non-specific interaction mayoccur between a molecule and a non-target cell or tissue. Nevertheless,specific binding can be distinguished as mediated through specificrecognition of the receptor or antigen. Specific binding results in amuch stronger association between the antibody (or other ligand) andcells bearing the antigen than between the bound antibody (or otherligand) and cells lacking the antigen. Specific binding typicallyresults in greater than 2-fold, preferably greater than 5-fold, morepreferably greater than 10-fold and most preferably greater than100-fold increase in amount of bound antibody or other ligand (per unittime) to a cell or tissue bearing IL-21 receptor as compared to a cellor tissue lacking IL-21 receptor. Specific binding to a protein undersuch conditions requires an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsare appropriate for selecting antibodies or other ligands specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow & Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork (1988), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity.

Substantially purified: The term substantially purified indicates thatthe subject is substantially free of other molecular or cellularconstituents with which it is naturally associated. Thus, asubstantially purified polypeptide as is a polypeptide that issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated, for example, in a cell.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated. A substantially purified population of cells (suchas B cells, B cell progenitors, mature B cells, memory B cells, plasmacells, etc.) is substantially free of other cellular components of thetissue in which it is naturally found, such as bone marrow, peripheralblood, spleen, lymph node, etc. For example, a substantially purepopulation of B cells (e.g., a B cell progenitor, an immature B cell, amature B cell, a memory B cell, a plasma cell, etc.) is at least 50%,for example at least about 80% or alternatively at least about 90% freeof other cellular components. In an embodiment, the population of Bcells is at least about 95% free of other cells. For example, apopulation of purified B cells, obtained from a tissue such asperipheral blood, is substantially free of red blood cells, T cells,platelets, and other cells typically found in peripheral blood.

Subject: Living multicellular vertebrate organisms, a category thatincludes both human and veterinary subjects for example, mammals, birdsand primates.

Supernatant: The culture medium in which a cell is grown. The culturemedium can include material from the cell, such as materials producedwithin the cell and/or secreted by the cell, such as cytokines andinterleukins.

Therapeutically Effective Amount: An amount sufficient to achieve adesired biological effect, for example an amount that is effective toinduce the differentiation of memory B or plasma cells. In particularexamples, it is an amount of an agent, such as IL-21, effective toinduce B cell differentiation in a subject, such as in a subject to whomit is administered (for example a subject with a deficiency of memory Bcells or plasma cells). In another particular example, a therapeuticallyeffective amount is an amount of IL-21 that alters a sign or a symptomof a disorder in a subject, such as a subject with a deficiency ofmemory B cells or plasma cells.

An effective amount of an agent such as IL-21 can be administered in asingle dose, or in several doses, for example daily, during a course oftreatment. However, the effective amount of IL-21 will be dependent onthe subject being treated, the severity and type of the condition beingtreated, and the manner of administration. The methods disclosed hereinhave equal application in medical and veterinary settings. Therefore,the general term “subject being treated” is understood to include allorganisms (e.g., humans, apes, dogs, cats, horses, and cows) thatrequire an increase in the desired biological effect, such as anenhanced immune response.

Transgene: A heterologous nucleic acid (“foreign gene”) that is placedinto an organism by introducing the nucleic acid into embryonic stem(ES) cells, newly fertilized eggs or early embryos. In one embodiment, atransgene is a polynucleotide sequence that encodes a polypeptide, forexample a sequence that encodes a marker polypeptide that can bedetected using methods known to one of skill in the art. In anotherembodiment, the transgene encodes a therapeutic polypeptide that can beused to alleviate or relieve a symptom of a disorder. In yet anotherembodiment, the transgene encodes a therapeutically effectiveoligonucleotide, for example an antisense oligonucleotide, whereinexpression of the oligonucleotide inhibits expression of a targetnucleic acid sequence. In a further embodiment, the transgene encodes anantisense nucleic acid or a ribozyme. In yet another embodiment, atransgene is a stop cassette.

In other embodiments, a transgene contains native regulatory sequencesoperably linked to the transgene (e.g., the wild-type promoter, foundoperably linked to the gene in a wild-type cell). Alternatively, aheterologous promoter can be operably linked to the transgene. In yetanother embodiment, a viral LTR can be used to express the transgene.

Transgenic Cell: Transformed cells that contain heterologous or foreign,DNA.

Transgenic Animal: An animal, for example, a non-human animal such as amouse, that has a heterologous nucleic acid or foreign gene introducedinto one or more of its cells. A transgene can be inserted into thegenome of an animal by random integration or by targeted insertion. Forexample, DNA can be integrated in a random fashion by injecting it intothe pronucleus of a fertilized ovum. In this case, the DNA can integrateanywhere in the genome, and multiple copies often integrate in ahead-to-tail fashion. There is no need for homology between the injectedDNA and the host genome.

Targeted insertion is accomplished by introducing the DNA into embryonicstem (ES) cells and selecting for cells in which the DNA has undergonerecombination with homologous genomic sequences. For this to occur,there often are several kilobases of sequence identity or similaritybetween the heterologous and genomic DNA, and positive selectablemarkers are often included. In addition, negative selectable markers areoften used to select against cells that have incorporated DNA bynon-homologous recombination (random insertion).

Vector: A means by which a nucleic acid molecule can be replicated ormanipulated, e.g., introduced into a cell. A vector can include nucleicacid sequences that permit it to replicate in the cell, such as anorigin of replication. A vector can also include one or more marker ortherapeutic transgenes and other genetic elements known in the art.Common vectors include viral vectors, phage vectors (e.g., adenoviralvectors, retroviral vectors, and Herpes viral vectors), bacterialvectors (such as plasmid vectors) and artificial chromosomes.

Wild-type: The term “wild-type” refers to a gene or gene product whichhas the characteristics of that gene or gene product when isolated froma naturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and/or functional properties (i.e. alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these aretypically identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

IL-21 Polypeptides and Polynucleotides

IL-21 polypeptides and polynucleotides encoding these polypeptides areused in the methods disclosed herein. A nucleic acid sequence andpolypeptide sequence for murine IL-21 is available at the NCBI websiteas GENBANK® Accession No. NM021782 and GENBANK® Accession No. AF254070.A nucleic acid and polypeptide sequence of human IL-21 is available atthe NCBI website as GENBANK® Accession No. NM021803 and GENBANK®Accession No. AF254069. These GENBANK® entries are incorporated byreference herein.

In one specific, non-limiting example, a human IL-21 polypeptide has thefollowing amino acid sequence

(SEQ ID NO: 1) MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKIHRLTCPSCDSYEKKPPKEFLERFKSL LQKMIHQHLSSRTHGSE.

In another specific, non-limiting example, a murine IL-21 has an aminoacid sequence set forth as

(SEQ ID NO: 2) MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKLKLPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS

Furthermore, sequence of human IL-21 is shown as SEQ ID NO: 1 in U.S.Published Patent Application No. 2003/0003545, which is incorporatedherein, by reference. A representative clone containing all or most ofthe sequence for IL-21 (designated HTGED19) was deposited with theAmerican Type Culture Collection (“ATCC”) on Mar. 5, 1998, and was giventhe ATCC Deposit Number 209666 (see, e.g., U.S. Published PatentApplication No. 2003/0003545).

IL-21 polypeptides (including variant polypeptides and IL-21 polypeptideanalogs, such as IL-21 agonists), can be composed of amino acids joinedto each other by peptide bonds or modified peptide bonds, such as,peptide isosteres, and can contain amino acids other than the 20gene-encoded amino acids. The IL-21 polypeptides can be modified byeither natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art.Modifications can occur anywhere in the IL-21 polypeptides, includingthe peptide backbone, the amino acid side-chains and the amino orcarboxyl termini. IL-21 polypeptides can be branched, for example, as aresult of ubiquitination, and they can be cyclic, with or withoutbranching. Cyclic, branched, and branched cyclic IL-21 polypeptides canresult from post-translation natural processes or can be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.

Variants of IL-21, or polynucleotides encoding these variants, can alsobe used in the methods disclosed herein. U.S. Published PatentApplication No. 2003/0003545 discloses polynucleotide or polypeptidediffering from the IL-21 polynucleotides or polypeptides, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the IL-21 polynucleotide orpolypeptide. For example, a variant of an IL-21 polynucleotide is apolynucleotide having a nucleotide sequence at least, for example, apolynucleotide can be at least 90% “identical” to a reference IL-21nucleotide sequence, such as at least 95%, 96%, 97%, 98% or 99%identical to a reference IL-21 sequence. Thus, the nucleotide sequenceof the polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence can include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence encoding theIL-21 polypeptide.

IL-21 variants have been described that contain alterations in thecoding regions, non-coding regions, or both. In one specific,non-limiting example, polynucleotide variants of IL-21 containalterations which produce silent substitutions, additions, or deletions,but do not alter the properties or activities of IL-21, such as avariant produced by the degeneracy of the genetic code. Moreover,variants in which 5-10, 1-5, or 1-2 amino acids are substituted,deleted, or added in any combination have been described (see, U.S.Published Patent Application No. 2003/0003545). Using known methods ofprotein engineering and recombinant DNA technology, variants can begenerated to improve or alter the characteristics of the IL-21polypeptides. For instance, one or more amino acids can be deleted fromthe N-terminus or C-terminus of the secreted protein without substantialloss of biological function (see, U.S. Published Patent Application No.2003/0003545). In several examples, one, two, three, four or five aminoacids are deleted from the N-terminus, the C-terminus, or both.

One of skill in the art can readily produce polynucleotide sequencesencoding this polypeptide, and can use genetic engineering to operablylink promoters to the polynucleotides sequences, and produce vectorsencoding this polypeptide using standard laboratory techniques (see, forexample, Sambrook et al., Molecular Cloning: a Laboratory Manual, ColdSpring Harbor Press, 1989).

Polynucleotides encoding an IL-21, including variants and analogs ofIL-21, such as IL-21 agonists and antagonists, are also of use in themethods disclosed herein. These polynucleotides include DNA, cDNA andRNA sequences which encode an IL-21 or a variant or analog thereof. Itis understood that all polynucleotides encoding an IL-21 are alsoincluded herein, as long as they encode a polypeptide that has anactivity of IL-21 (that is, are agonist polypeptides), such as theability to induce apoptosis of a B cell. Such polynucleotides includenaturally occurring, synthetic, and intentionally manipulatedpolynucleotides. For example, a polynucleotide encoding IL-21 can besubjected to site-directed mutagenesis. The polynucleotides includesequences that are degenerate as a result of the genetic code, butencode IL-21. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are of use in the methods disclosed herein as long as theamino acid sequence of the IL-21 encoded by the nucleotide sequence isfunctionally unchanged.

DNA sequences encoding an IL-21 can be expressed in vitro by DNAtransfer into a suitable host cell. Methods of stable transfer, meaningthat the foreign DNA is continuously maintained in the host, are knownin the art.

Polynucleotide sequences encoding an IL-21, including IL-21 variants andanalogs, can be inserted into an expression vector, such as a plasmid,virus or other vehicle known in the art that has been manipulated byinsertion or incorporation of the IL-21 sequences. Polynucleotidesequences which encode an IL-21 can be operatively linked to expressioncontrol sequences. In one embodiment, an expression control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with theexpression control sequences.

The polynucleotide encoding an IL-21, such as human IL-21, can beinserted into an expression vector that contains a promoter sequencewhich facilitates the efficient transcription of the inserted geneticsequence by the host. The expression vector typically contains an originof replication, a promoter, as well as specific genes that allowphenotypic selection of the transformed cells. Vectors suitable for useinclude, but are not limited to, the T7-based expression vector forexpression in bacteria (Rosenberg et al., Gene 56:125, 1987), the pMSXNDexpression vector for expression in mammalian cells (Lee and Nathans, J.Biol. Chem. 263:3521, 1988) and baculovirus-derived vectors forexpression in insect cells. The DNA segment can be present in the vectoroperably linked to regulatory elements, for example, a promoter (e.g.,T7, metallothionein I, or polyhedrin promoters).

Polynucleotide sequences encoding IL-21 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.For example, biologically functional viral and plasmid DNA vectorscapable of expression and replication in a host are known in the art.Such vectors are used to incorporate a DNA sequence encoding an IL-21.Transfection of a host cell with recombinant DNA can be carried out byconventional techniques and are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, methods of transfection of DNA as calciumphosphate co-precipitates, conventional mechanical procedures such asmicroinjection, electroporation, insertion of a plasmid encased inliposomes, or virus vectors can be used. Eukaryotic cells can also becotransformed with a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed IL-21 polypeptide, orfragments thereof, can be carried out by conventional means includingpreparative chromatography, affinity column chromatography, andimmunological separations involving monoclonal or polyclonal antibodies,and used in the methods disclosed herein.

Methods of Inducing B Cell Differentiation

Methods are provided herein for expanding and/or inducingdifferentiation of a B cell progenitor, (such as an immature B cell),and/or a mature naïve or memory B cell into a memory B cell or a plasmacell. For example, the methods include inducing differentiation of a Bcell progenitor or a naïve mature B cell into a memory cell or a plasmacell. The methods also include inducing differentiation of a memory cellinto a plasma cell, as well as inducing expansion of memory cells. Themethods involve contacting an immature, naïve or memory B cell with atherapeutically effective amount of an agent that activates JAK1, JAK3,STAT5A or STAT5B and isolating a plasma cell and/or a memory cell,thereby producing differentiated plasma cells and/or memory B cells. Inone embodiment, the agent is an IL-21 polypeptide or an IL-21 agonist.The B cell progenitors (e.g., immature B cells, such as transitional Bcells), naïve B cells and/or memory B cells can be included in a mixedpopulation of cells, or can be a purified or enriched populations ofcells. The cells can be from any mammal. In one example, the cells arehuman cells.

In one embodiment, a B cell progenitor (e.g., an immature B cell, suchas a transitional B cell), naïve B cell, and/or memory cell is contactedwith IL-21 or an IL-21 agonist in vitro. Thus, in order to inducedifferentiation, a population of cells including the B cell progenitor,naïve B cell and/or memory B cell is isolated from a subject and thencontacted with an effective amount of IL-21 polypeptide or an agonistthereof. The immature B cells, naïve mature B cells and/or memory cellscan be purified populations of cells, or can be included in a mixedpopulation of cells that are isolated from the subject, such as bonemarrow derived cells, cord blood cells or peripheral blood cells.

In another embodiment, a population of cells including immature, naïveor memory B cells is obtained from a subject, contacted with atherapeutically effective amount of IL-21 or an agonist thereof, andplasma cells and/or memory cells are isolated. Typically, asubstantially pure population of plasma cells and/or memory B cells areisolated. Optionally, a mixed population is isolated. Typically, where amixed population of cells is isolated, it is enriched for plasma cellsand/or memory B cells. The isolated plasma cells and/or memory cells arethen introduced into the same subject (autologous) or another subject(heterologous).

In another embodiment, in order to enhance an immune response, apopulation of cells including a B cell progenitor, (such as an immatureB cell or transitional B cell), a naïve B cell, or a memory B cell iscontacted with IL-21 or an agonist thereof. Memory B cells and/or plasmacells are isolated and the memory B cells and/or the plasma cells areintroduced into a subject. The subject can be any mammalian subject,such as, but not limited to, a human subject. The population of cellsincluding the B cell progenitor, naïve mature B cell or memory cell canbe further contacted with an antigen. The antigen can be any antigen ofinterest, including, but not limited to, antigens from a virus,bacteria, or parasite. In one embodiment, the B cell progenitors, naïveB cells or memory B cells are contacted with IL-21 and an antigen,either sequentially or simultaneously.

Fluorescence activated cell sorting (FACS) can be used to sort (isolate)cells, such as various populations of immature and mature B cells ordifferentiated plasma cells or memory cells, by contacting the cellswith an appropriately labeled antibody. In one embodiment, severalantibodies (such as antibodies that bind CD19, IgD, CD38, CD27 and/orIgM) and FACS sorting can be used to produce substantially purifiedpopulations of immature B cells, plasma cells and or memory B cells.These methods are known in the art, and exemplary protocols aredescribed below.

A FACS employs a plurality of color channels, low angle and obtuselight-scattering detection channels, and impedance channels, among othermore sophisticated levels of detection, to separate or sort cells. AnyFACS technique can be employed as long as it is not detrimental to theviability of the desired cells. (For exemplary methods of FACS see U.S.Pat. No. 5,061,620).

However, other techniques of differing efficacy can be employed topurify and isolate desired populations of cells. The separationtechniques employed should maximize the retention of viability of thefraction of the cells to be collected. The particular technique employedwill, of course, depend upon the efficiency of separation, cytotoxicityof the method, the ease and speed of separation, and what equipmentand/or technical skill is required.

Separation procedures include magnetic separation, using antibody-coatedmagnetic beads, affinity chromatography, cytotoxic agents, either joinedto a monoclonal antibody or used in conjunction with complement, and“panning,” which utilizes a monoclonal antibody attached to a solidmatrix, or another convenient technique. Antibodies attached to magneticbeads and other solid matrices, such as agarose beads, polystyrenebeads, hollow fiber membranes and plastic petri dishes, allow for directseparation. Cells that are bound by the antibody can be removed from thecell suspension by simply physically separating the solid support fromthe cell suspension. The exact conditions and duration of incubation ofthe cells with the solid phase-linked antibodies will depend uponseveral factors specific to the system employed. The selection ofappropriate conditions, however, is well within the skill in the art.

The unbound cells then can be eluted or washed away with physiologicbuffer after sufficient time has been allowed for the cells expressing amarker of interest (e.g., CD45) to bind to the solid-phase linkedantibodies. The bound cells are then separated from the solid phase byany appropriate method, depending mainly upon the nature of the solidphase and the antibody employed.

Antibodies can be conjugated to biotin, which then can be removed withavidin or streptavidin bound to a support, or fluorochromes, which canbe used with a fluorescence activated cell sorter (FACS), to enable cellseparation.

For example, cells expressing CD45 are initially separated from othercells by the cell-surface expression of CD45. In one specific,non-limiting example, CD45⁺ cells are positively selected by magneticbead separation, wherein magnetic beads are coated with CD45 reactivemonoclonal antibody. The CD45⁺ cells are then removed from the magneticbeads.

Release of the CD45⁺ cells from the magnetic beads can effected byculture release or other methods. Purity of the isolated CD45⁺ cells isthen checked with a FACSCAN®. flow cytometer (Becton Dickinson, SanJose, Calif.), for example, if so desired. In one embodiment, furtherpurification steps are performed, such as FACS sorting the population ofcells released from the magnetic beads. In one example, this sorting canbe performed to detect expression of CD19, CD44, CD21, CD62L, and CD10to detect or isolate immature B cells. In another example, mature Bcells can be isolated and/or detected on the basis of expression ofCD19, IgD, CD38, CD27, CD21, and/or CD23.

In a further specific, non-limiting example, the immature B cell is invivo. Thus, a therapeutically effective amount of IL-21 or an agonistthereof is administered to the subject, in order to inducedifferentiation of the immature B cell into a plasma cell or a memorycell. The subject can be any subject of interest, including subjectswith a deficiency of memory B cells, plasma cells, or both. In oneembodiment, the subject has a specific immunodeficiency, such as adeficiency that results in a reduction in number or function of memory Bcells, and or plasma cells. In one example, the subject has a B celldeficiency after bone marrow transplantation, e.g., after allogeneicbone marrow transplantation. In other examples, the subject hastransient hypgammaglobulinemia of childhood, Hyper-IgM syndrome,X-linked agammagloulinemia, common variable immunodeficiency, ataxiateleangiectasia, or a selective IgA or IgG subclass deficiency.

An IL-21 polypeptide or an agonist thereof can be administered by anymeans known to one of skill in the art (see Banga, “ParenteralControlled Delivery of Therapeutic Peptides and Proteins,” inTherapeutic Peptides and Proteins, Technomic Publishing Co., Inc.,Lancaster, Pa., 1995) such as by intramuscular, subcutaneous, orintravenous injection, but even oral, nasal, or anal administration iscontemplated. In one embodiment, administration is by subcutaneous orintravascular injection. To extend the time during which the peptide orprotein is available to stimulate a response, the peptide or protein canbe provided as an implant, an oily injection, or as a particulatesystem. The particulate system can be a microparticle, a microcapsule, amicrosphere, a nanocapsule, or similar particle. (see, e.g., Banga,supra).

Nucleic acid based therapy to induce the production of memory B cells orplasma cells is also disclosed herein. Such therapy would achieve itstherapeutic effect by introduction of a therapeutically effective amountof a polynucleotide encoding the IL-21 into a subject to achieveexpression of IL-21. Without being bound by theory, IL-21 expressedfollowing treatment with a therapeutic polynucleotide inducesdifferentiation of an immature B cell into memory B cells and/or plasmacells. Delivery of the therapeutic polynucleotide can be achieved usinga recombinant expression vector such as a chimeric virus or a colloidaldispersion system, or targeted liposomes.

Various viral vectors which can be utilized for nucleic acid basedtherapy as taught herein include adenovirus or adeno-associated virus,herpes virus, vaccinia, or an RNA virus such as a retrovirus. In oneembodiment, the retroviral vector is a derivative of a murine or avianretrovirus, or a human or primate lentivirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). In one embodiment, when the subject is a human, avector such as the gibbon ape leukemia virus (GaLV) is utilized. Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated. Byinserting a nucleic acid encoding IL-21 into the viral vector, alongwith another gene which encodes the ligand for a receptor on a specifictarget cell, for example, the vector is now target specific. Retroviralvectors can be made target specific by attaching, for example, a sugar,a glycolipid, or a protein. In one specific, non-limiting example,targeting is accomplished by using an antibody to target the retroviralvector.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to, Q2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for a polynucleotide encoding IL-21 isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. One colloidal dispersion system is a liposome.Liposomes are artificial membrane vesicles which are useful as deliveryvehicles in vitro and in vivo. It has been shown that large unilamellarvesicles (LUV), which range in size from 0.2-4.0 microns, canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley et al., Trends Biochem. Sci. 6:77, 1981). Inaddition to mammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the nucleic acidof interest at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino etal., Biotechniques 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids can also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidyl-glycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include, for example, phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticuloendothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system can be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

For administration to a subject, a therapeutically effective dose of apharmaceutical composition containing nucleic acid encoding IL-21, anIL-21 polypeptide or an IL-21 agonist, can be included in apharmaceutically acceptable carrier. Optionally, other agents can beincluded in the pharmaceutical composition. For example, an antigen canalso be administered in conjunction with the nucleic acid encodingIL-21, IL-21 polypeptide, or IL-21 agonist, either simultaneously orsequentially. In another example, an additional cytokine can beincluded.

The pharmaceutical compositions are prepared and administered in doseunits. Solid dose units are tablets, capsules and suppositories. Fortreatment of a patient, depending on activity of the compound, manner ofadministration, nature and severity of the disorder, age and body weightof the patient, different daily doses are necessary. Under certaincircumstances, however, higher or lower daily doses can be appropriate.The administration of the daily dose can be carried out both by singleadministration in the form of an individual dose unit or else severalsmaller dose units and also by multiple administrations of subdivideddoses at specific intervals.

The pharmaceutical compositions are in general administered topically,intravenously, subcutaneously, intramuscularly, orally or parenterallyor as implants, but even rectal use is possible in principle. Suitablesolid or liquid pharmaceutical preparation forms are, for example,granules, powders, tablets, coated tablets, (micro)capsules,suppositories, syrups, emulsions, suspensions, creams, aerosols, dropsor injectable solution in ampoule form and also preparations withprotracted release of active compounds, in whose preparation excipientsand additives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, flavorings, sweeteners orsolubilizers are customarily used as described above. The pharmaceuticalcompositions are suitable for use in a variety of drug delivery systems.For a brief review of present methods for drug delivery, see Langer,Science 249:1527-1533, 1990.

A therapeutically effective dose of a pharmaceutical compositioncontaining nucleic acid encoding IL-21, or an IL-21 polypeptide, can bedelivered locally or systemically. A therapeutically effective dose isthe quantity of an IL-21, or a nucleic acid encoding an IL-21, thatcures or at least partially arrests the symptoms of the deficiency ofmemory B cells or plasma cells. Amounts effective for this use will, ofcourse, depend on the severity of the disease and the weight and generalstate of the patient. Typically, dosages used in vitro can provideuseful guidance in the amounts useful for in situ administration of thepharmaceutical composition, and animal models can be used to determineeffective dosages for treatment of particular disorders. Variousconsiderations are described, e.g., in Gilman et al., eds., Goodman AndGilman's: The Pharmacological Bases of Therapeutics, 8th ed., PergamonPress, 1990; and Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Co., Easton, Pa., 1990, each of which is herein incorporatedby reference.

Screening

A method is disclosed herein for identifying an agent with aphysiological effect on differentiation of a memory B cells and/or aplasma cell. The method includes contacting a population of cellsincluding B cell progenitors, such as substantially purified immature Bcells, naïve B cells or memory cells with an effective amount of IL-21and an agent of interest, and determining the effect of the agent onmemory B cell and/or plasma cell differentiation. For example, theeffect on differentiation of plasma cells can be evaluated by measuringantibody production, or by detecting one or more markers thatcharacterize plasma cells. Similarly, the effect on production of memorycells can be evaluated using one or more markers that characterizememory cells. In one embodiment, the agent inhibits the differentiationof the memory B cell and/or the plasma cell as compared to a control. Inanother embodiment, the agent stimulates the differentiation of thememory B cells/and or the plasma cell as compared to a control.

Suitable controls include populations of cells not contacted with IL-21and not contacted with the agent, cells contacted with IL-21 in theabsence of the agent, or a standard value. Agents identified by theassay can be selected for further study if, for example, they show astatistically different result from a control. For example, a student'sT-test is used to compare the values obtained in the assay with thecontrol values. A statistically significant result is then considered tobe one in which p<0.05.

The agent can be any agent of interest. In one embodiment, therapeuticagents that are derived from combinatorial chemical libraries, arescreened in high throughput assays. Agents identified from the libraryare further characterized and/or detected using appropriate methods.

In the event that the agent is a nucleic acid, any of a variety ofprocedures can be used to further characterize the nucleic acid (such asa gene of interest). For example, nucleic acid agents identified fromthe library are further evaluated, detected, cloned, sequenced, and thelike, either in solution or after binding to a solid support, by anymethod usually applied to the detection of a specific DNA sequence, suchas PCR, oligomer restriction (Saiki et al., Biol. Technology3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis(Conner et al., Proc. Natl. Acad. Sci. USA 80:278, 1983),oligonucleotide ligation assays (OLAs) (Landegren et al., Science241:1077, 1988), and the like. In addition, any of a variety ofprocedures can be used to clone genes of interest when the testcomposition is expressed as a gene product in a combinatorial library(as opposed to a chemical composition). One such method entailsanalyzing a shuttle vector library of DNA inserts (derived from a cellwhich expresses the composition) for the presence of an insert whichcontains the gene. For example, cells are transfected with the vector,and then assayed for expression of the product of interest. Thepreferred method for cloning these genes entails determining the aminoacid sequence of the composition protein, for example by purifying thedesired protein and analyzing it with automated sequencers.

The proteins can be extracted and purified from the culture media or acell by using known protein purification techniques, such as extraction,precipitation, ion exchange chromatography, affinity chromatography, gelfiltration and the like. The therapeutic proteins can be isolated byaffinity chromatography.

A method is also disclosed herein for identifying an agent that inhibitsan activity of IL-21. The method includes contacting a cell with anagent and determining if the agent inhibits Bcl-6, Blimp-1 and/or AID.The identification of an agent as inhibiting Bcl-6, Blimp-1 and/or AIDidentifies the agent being of use to inhibit the activity of IL-21. Inone embodiment, the agent inhibits one or more of Bcl-6, Blimp-1 and AIDas compared to a control. Suitable controls are cells not contacted withthe agent, such as cells contacted with an agent that is known not to bean IL-21 receptor antagonist, or a cell contacted with a buffer in theabsence of a test agent. Suitable controls also include untreated cellsor a standard value.

Bcl-6 is described in U.S. Pat. No. 6,174,997, herein incorporated byreference. In one embodiment, the agent is an antisense molecule thatspecifically binds Bcl-6. Exemplary antisense molecules are described,for example, in U.S. Pat. No. 6,140,125. The agent can also be anantibody that specifically binds Bcl-6. Exemplary antibodies aredescribed in U.S. Pat. No. 5,882,858.

Blimp-1 is described in U.S. Pat. No. 6,586,579, herein incorporated byreference. In one embodiment, the agent is an antisense molecule thatspecifically binds a nucleic acid encoding Blimp-1, or an antibody thatspecifically binds Blimp-1 protein. Thus, as disclosed herein, theseantisense agents or antibodies can be used to inhibit an activity ofIL-21. Similarly, antisense molecules that bind specifically to anucleic acid encoding AID, or an antibody that specifically binds AIDprotein can be utilized to inhibit an activity of IL-21.

As discussed above, the agent can be any agent of interest. The agentcan be a chemical compound, nucleic acid, antibody, or any smallmolecule that affects transcription of Bcl-6, Blimp-1 and/or AID, oraffects an activity of Bcl-6 polypeptide, or Blimp-1 polypeptide and/orAID polypeptide. As described above, upon detection an agent can befurther characterized by suitable methods.

Methods of detecting the inhibition of Blimp-1 or Bcl-6 expressionand/or are known in the art. For example, the expression of Blimp-1 orBcl-6 can be analyzed by detecting the presence of mRNA encoding Blimp-1or Bcl-6. For example, Blimp-1 expression can be evaluated by detectingan RNA encoding Blimp-1 exemplified by the sequences represented byGENBANK® Accession Nos. U08185 or AF084199, or a homolog thereof. Bcl-6expression can be evaluated by detecting an RNA encoding Bcl-6exemplified by the sequences represented by GENBANK® Accession Nos.NM009744 or U00115, or a homolog thereof. Similarly, AID expression canbe evaluated by detecting an RNA encoding AID, exemplified by sequencesrepresented by GENBANK® Accession Nos. ABA40430 and ABA40431. One ofskill in the art can readily measure the presence of a specific mRNA,using techniques such as, but not limited to Northern blot, Dot blots,reverse transcriptase polymerase chain reaction (RT-PCR), or real-timePCR. The expression of Blimp-1 or Bcl-6 can also be analyzed bydetecting the presence of Blimp-1 or Bcl-6 proteins. Exemplary Blimp-1proteins that can be detected include those represented by GENBANK®Accession Nos. AAA19252, AAC33300, and homologs thereof. Exemplary Bcl-6proteins that can be detected include those represented by GENBANK®Accession Nos. AAB17432, AAC50054, and homologs thereof. For example,assays such as immunohistochemical assays, radioimmune assays, or ELISAassays can be used to detect Blimp-1 or Bcl-6 proteins.

An activity of Blimp-1 or Bcl-6 can also be measured. For example,Blimp-1 or Bcl-6 DNA binding activity can be measured using assays knownto those of skill in the art, such as, but not limited to,electrophoretic mobility shift assays. If an agent decreases the amountof Bcl-6 or Blimp-1 mRNA, decreases the amount of Bcl-6 or Blimp-1protein, or decreases the activity of Blimp-1 or Bcl-6 as compared to acontrol, then the agent is of use in inhibiting an activity of IL-21.One of skill in the art can readily determine a significant decrease inthese parameters, such as a 25%, a 50%, a 75%, 80%, 85%, 90%, 95%, or99% decrease, using standard statistical methods. Similarly, a decreasein the expression or activity of AID can also be measured by those ofskill in the art.

Hybridoma Technology

Although numerous protocols, reagents and fusion partners are availablefor producing mouse B cell hybridomas, there are very limited resourcesavailable for producing monoclonal antibodies from human B cells. Forexample, there are relatively few human B cell fusion partners (e.g.,LICR-2 (HMY2), HK-128, and SPAZ-4 cells), which can be successfullyfused to non-transformed B cells, although these fusion partners can befused to EBV-transformed or malignant human B cells. In addition, humanB cells have been differentiated into plama cells and plasma blasts,especially of known specificity, only with low efficiency. Mitogens suchas pokeweed mitogen or PMA have been used to activate human B cells inthe presence of various cytokine cocktails in attempts to generatestimulated human B cells that will fuse but this has met with limitedsuccess. Activated T cells have also been used in attempts to generate Bcell hybridomas (Bergman et al., J. Immunology 156:3118-3132, 1996).

IL-21 not only induces human B cells to intensely proliferate (which isrequired for successful fusion of hybridomas) but also induce thedifferentiation of human naïve and memory B cells intoantibody-producing plasma cells. Thus, IL-21 can be used to activate Bcells (e.g., in combination with anti-CD40 and/or anti-IgM andanti-CD40) to generate B cells that will successfully fuse with theavailable fusion partners. For example, naïve human B cells can beprimed in vitro. Alternatively, naïve human B cells can be transferredinto NOD/SCID mice, which are then immunized in the presence of solubleT cell co-stimulators, followed by in vitro co-activation with IL-21,and optionally with additional stimulatory agents, such as anti-CD40and/or anti-IgM. Following in vitro or in vivo priming, the human bcells can be fused with partners, such as those selected form the celllines listed above, to generate human B cell hybridomas of knownspecificities. In addition, screening of libraries of hybridomasproduced by fusing memory B cells co-activated with IL-21 (andoptionally with anti-CD40) can be used to identify human B cellhybridomas of know specificities.

EXAMPLES

The disclosure is illustrated by the following non-limiting Examples.

Example 1 Materials and Methods

Mouse Splenic B cell preparation and proliferation assays. C57BL/6 micewere obtained from Jackson Laboratory and (C57BU6×129) F1 mice weregenerated at NIH. Splenic B cells were isolated using B220- orCD43-magnetic beads (Miltenyi) and were >95% pure as assessed by flowcytometry using B220-PE and TCRβ-APC. B cells isolated from γ_(c) KOmice (which have diminished B cell numbers), were approximately 90%pure. B cells were plated in 96 well plates at 10⁵ cells/well and weretreated as indicated with LPS (5 μg/ml, Sigma), anti-CD40 (1 μg/ml, BDPharMingen), anti-IgM (5 μg or 2.5 μg/ml; Sigma or JacksonImmunolaboratory), murine IL-4 (200 U/ml), and murine IL-21 (50 ng/ml).For proliferation assays, cells were cultured for either two or fourdays and pulsed with ³H-thymidine (1 μCi/well) for the last 18 hours ofculture.

Isolation of human B cells. Human peripheral B cells were isolated frombuffy coats of anonymous healthy donors drawn at the National Institutesof Health Division of Transfusion Medicine. Umbilical cord blood sampleswere obtained from Advanced Bioscience Resources, Inc. (Alameda,Calif.). Generally, human cord blood and peripheral B cells wereisolated by negative selection using the Rosette-sep technique followingthe manufacturer's instructions (Stem Cell Technology, Vancouver,British Columbia). Preparations were typically >96% CD19⁺ fromperipheral blood. Negatively-selected cord blood B cell isolationsyielded more heterogeneous cell populations. Therefore, B cells werepositively-selected with anti-CD19 magnetic beads (Miltenyi Biotech,Auburn, Calif.) in some of the cord blood experiments and these weretypically >95% pure. For further purity of peripheral B cells, CD20⁺CD27⁻ naïve B cells or CD20⁺ CD27⁺ memory B cells were isolated on aMoFlo cell sorter, (Dako Cytomation, Carpinteria, Calif.). Briefly,negatively-selected B cells were incubated at 10⁷ cells/ml in stainingbuffer with mAbs specific for CD20 and CD27 and in some experimentsanti-IgD as well. Cells were incubated for 30 min at 4° C., washed andsorted into CD20⁺ CD27⁺ or CD20⁺ CD27⁻ subsets. Preparations weretypically >98% pure.

Human B cell culture, activation and CFSE labeling. Purified B cellswere cultured at 1×10⁶ cells/ml in either 1 ml in 24 well culture platesor 100 μl in 96 well round bottom culture plates. The cells wereincubated with a combination of human IL-2 (100 U/ml, Roche,Indianapolis, Ind.), human IL-4 (100 ng/ml, R&D systems, Minneapolis,Minn.), human IL-21 (100 ng/ml, R&D systems), human IL-10 (25 ng/ml, R&Dsystems) and either 1 μg/ml anti-human CD40 (R&D systems), 5 μg/mlanti-IgM (Jackson ImmunoResearch Laboratories, (West Grove, Pa.), or0.01% final dilution of heat killed, formalin-fixed SAC (Calbiochem, LaJolla, Calif.). In some experiments, purified B cells were labeled withCFSE. In brief, purified B cells were washed extensively in PBS toremove all FCS, and CFSE/PBS (Molecular Probes, Eugene, Oreg.) was addedat a final concentration of 2.5 μM to 2-5×10⁷ cells/ml for 8 minutes.Labeling was quenched by addition of FCS, and cells were washed fourtimes in medium containing 10% FCS before culture. To obtain an accuratecomparison for the controls, cord blood B cells were stimulated in thepresence of IL-4 which facilitated cell survival but induced neither IgDdown-modulation or plasma cell differentiation.

Human B cell proliferation assays. To assess proliferative responses ofcultured cells, 10⁵ purified B cells were cultured as described above in96 well round bottom plates. After 3-5 days of culture, ³[H]-thymidine(37 Kbq/well) was added to the cultures for an additional 16 hours.Thymidine uptake was measured using a liquid scintillation counter.

Western blotting. Clarified whole cell lysates were subjected toSDS-PAGE and Western blotting using anti-PARP (Cell SignalingTechnology, MA). For Blimp-1, whole cell extracts (20 μg) werefractionated on 8% SDS-gels (Invitrogen) and transferred to Immobilon Pmembranes (Millipore). After blocking in 5% milk/TBST, blots wereincubated overnight in rabbit anti-Blimp-1, washed, and incubated inHRP-labeled goat anti-rabbit IgG. Blots were development with anenhanced chemiluminescent substrate (Amersham).

Staining for apoptotic cells. Apoptosis was assessed using annexin-V and7-AAD (BD PharMingen, CA) and the TUNEL staining reagent (Roche AppliedScience, IN). Staining was performed according to the manufacturer'sinstructions.

Transgenic and knockout mice. Murine or human IL-21 cDNA constructscontaining V5 and His tags were generated by PCR and inserted into pHSE,a plasmid in which the expressed cDNA is under the control of theH-2k^(b) promoter and IgM enhancer (Pircher, EMBO J. 8:719-727, 1989;Held, J. Exp. Med. 184:2037-2041, 1996). The plasmid was linearized withXho1, DNA purified, and 2 ng was microinjected into the pronuclei offertilized oocytes from superovulated female C57BL/6×CBA F1/J mice andtwo days later implanted into the oviducts of pseudopregnant fostermothers. The founders were interbred with wild-type littermates.IL-21R^(−/−) mice were generated as previously described (Ozaki et al.Science 298:1630-1634, 2002).

In vivo transient expression of IL-21. The murine IL-21 cDNA wassubcloned into the pORF expression vector (InvivoGen, San Diego,Calif.), and 20 μg of DNA in 2 ml saline was injected intravenously intoC57BL/6 mice within 5 seconds (hydrodynamics-based transfection) (Liu etal., Gene Therapy 6:1258-1266, 1999; Zhang et al., Human Gene Therapy10:1735-1737, 1999). At days 4 and 7, the mice were analyzed with eithersaline or pORF-injected mice as control.

Flow cytometric analysis of mouse B cell populations. Cell populationswere stained with the following commercially obtained antibodies: FITCanti-CD21, CD23 and IgM, PE anti-CD23 and IgD, APC anti-B220 (BDPhanmingen). Staining with and AA4.1 antibodies was revealed with eitherPE- or Cy-conjugated streptavidin (BD Pharmingen). Analyses wereconducted using a FACSCaliber flow cytometer and data was analyzed usingCellQuest software (BD Immunosystems).

Flow Cytometric analysis of human B cell populations. Four color flowcytometry was performed using a FACSCalibur (Becton Dickinson, Calif.).Briefly, supernatants were collected and then all cells were harvestedfrom 96 well cultures at the end of the incubation period and stainedfor 30 minutes on ice with a combination of mAb obtained from BDBiosciences (Palo Alto, Calif.). The combination of anti-IgD-FITC,CD27-PE, anti-CD19-PerCP-Cy5.5 and anti-CD38-APC (clone HB7) wasroutinely used. Viable cells were identified by gating on lymphocytesand cells were analyzed immediately. All samples were collected for 1minutes, and as a result the density of the dot plots reveals relativecell numbers. In some experiments, to obtain total B cell numbers moreaccurately, AccuCount Particles (Spherotech, Libertyville, Ill.) wereadded before analyzing samples by flow cytometry and total cell numberswas determined as per manufacture's instructions. In some experiments,the combination of CFSE, anti-IgD-PE, anti-CD19 PerCP-Cy5.5, andanti-CD38-APC was used.

Determination of human Ig Levels. Secreted Ig in the culture supernatantof human B cells was quantitated by ELISA. Briefly, 96-well flat-bottomNunc-Brand Immuno plates (Nalge Nunc International, Rochester, N.Y.)were coated with 5 μg/mL of either affinity purified goat anti-human IgMor goat anti-human IgG-Fc (Bethyl Laboratories, Montgomery, Tex.)overnight at 4° C. Wells were then washed and blocked with a 0.2%BSA/PBS, and then the culture supernatant were titered onto the treatedplates and incubated overnight at 4° C. Bound Ig was detected with 0.5μg/ml alkaline phosphatase conjugated goat anti-human IgM or IgG (BethylLaboratories) and developed with p-nitrophenyl phosphate tablets(Sigma-Aldrich Inc, St. Louis, Mo.). Specific absorbance was measuredand optical density quantified at 410 nM by a Powerwave X 96-well platereader (Bio-tek Instruments, Inc, Winooski, Vt.). IgE concentrationswere measured with the Human IgE ELISA kit (Bethyl Laboratories).Specific IgG isotypes were differentiated using the Human IgG SubclassProfile ELISA KIT (Zymed Laboratories, Inc., San Francisco, Calif.).

Immunohistochemical staining of lymphoid follicles. Mouse spleens wereremoved and subdivided for analysis by either immunohistology or flowcytometry. For immunohistology, tissues were embedded inTissue-Tek/O.C.T. compound (Sakura, Zoeterwoude, the Netherlands),frozen in liquid nitrogen, and serially sectioned. Frozen tissuesections were immediately fixed in ice-cold acetone for 5 minutes andstained for 45 minutes in a humid chamber with either biotinylatedMAdCAM-1 (Southern Biotech), rat antibody supernatant specific for IgD(clone 1126C), or purified rat antibody specific for MCA1849 (MARCO,Serotec, Raleigh, N.C.). The sections were washed and bound antibodieswere revealed with either SA-conjugated or goat anti-rat conjugatedOregon Green (Molecular Probes, Eugene, Oreg.). IgM was detected withdirectly conjugated goat anti-mouse IgM Texas Red (SouthernBiotechnology).

Electrophoretic Mobility Shift Assays. Nuclear extracts were preparedfrom splenic B cells cultured with anti-IgM±IL-21 for 24 hours. Five μgwere used for DNA binding reactions with either a Blimp-1 binding site(MHC2TA) from the class II MHC promoter (Piskurich et al., NatureImmunol. 1:526-532, 2000) or with a Bcl-6 consensus binding site (Relijcet al., J. Exp. Med. 192:1841-1847, 2000). The double strandedoligonucleotides were as follows (only top strand is shown):

MHC2TA 5′-CAGTCCACAGTAAGGAAGTGAAATTAATTT-3′ (SEQ ID NO: 3) Bcl-65′-GAAAATTCCTAGAAAGCATA-3′ (SEQ ID NO: 4)

Real time PCR. Blimp-1, Bcl-6 and Pax5 mRNA levels in murine B cellpopulations were quantitated relative to GAPDH RNA levels by real timePCR. RNA was reverse transcribed using an Omniscript kit (Qiagen)according to the manufacturer's directions and PCR was performed using aQuantitect Probe Detection system (Qiagen). The oligonucleotides usedfor this study were:

Blimp-1

FW 5′-ACAGAGGCCGAGTTTGAAGAGA-3′ (SEQ ID NO: 5) RV5′-AAGGATGCCTCGGCTTGAA-3′ (SEQ ID NO: 6) TP5′-[6-FAM]CCCTGGGATTCCGGCGCTG[TA (SEQ ID NO: 7) MRA-6-FAM]-3′PAX5

FW 5′-AAACGCAAGAGGGATGAAGGT-3′ (SEQ ID NO: 8) RV5′-AACAGGTCTCCCCGCATCT-3′ (SEQ ID NO: 9) TP5′-[6-FAM]CACTTCCGGGCCGGGACTTC (SEQ ID NO: 10) C[TAMRA-6-FAM]-3′Bcl-6

FW 5′-TCAGAGTATTCGGATTCTAGCTGT (SEQ ID NO: 11) GA-3′ RV5′-TGCAGCGTGTGCCTCTTG-3′ (SEQ ID NO: 12) TP5′-[6-FAM]TGCAACGAATGTGACTGCCGT (SEQ ID NO: 13) TTCTCT[TAMRA-6-FAM]-3′GAPDH

FW 5′-TTCACCACCATGGAGAAGGC-3′ (SEQ ID NO 14) RV5′-GGCATGGACTGTGGTCATGA-3′ (SEQ ID NO: 15) TP5′-[6-FAM]TGCATCCTGCACCACCAACTG (SEQ ID NO: 16) CTTAG[TAMRA-6-FAM]-3′

Blimp-1, Bcl6, AICDA, PAX-5 and β-2 microglobulin mRNA levels wereevaluated by real time PCR in negatively or positively purified human Bcells. After 3 days in culture, cells were harvested and resuspended inTRIzol (Invitrogen, Carlsbad, Calif.) and stored at −70° C. RNA wasisolated using the RNeasy mini kit (Qiagen, Valencia, Calif.). Puritywas measured using spectrophotometry. Reverse transcription reactionswere prepared using the Superscipt One-Step PCR System with Platinum TaqPolymerase® and ROX reference dye (Invitrogen). 50 ng isolated RNA wasadded per reaction with 1.2 mM MgSO₄. Taqman Assays-on Demand Geneexpression primer/probe sets (Applied Biosystems) were used for Blimp-1(Hs00153357_ml), BC16 (HsO0153368_ml), AICDA (HsO0221068_ml), PAX-5(HsO0277134_ml) and β-2 microglobulin (β-2M) (Hs99999907_ml). Finalconcentrations were 1.8 μM for primers and 0.5 μM for probes. RT PCR wasperformed using the ABI Prism® 7700 Sequence Detection System and cycleconditions and relative quantification were completed as described bymanufacturers' instructions (Applied Bio Sysytems, Foster City, Calif.).Expression of each transcription factor was calculated using theComparative CT method with efficiency calculations and with all mRNAlevels normalized to β-2M. All reported values were then furthernormalized to control conditions, usually IL-2 or IL-2 and IL-4 (withoutany stimuli) as a value of 1.

Somatic Hypermutation: Human cord blood B cells were stimulated withIL-2 and IL-21 in the presence of anti-CD40 with or without anti-IgM for7-12 days. Individual live CD19⁺, IgD⁻ CD38⁺ class-switched B cells andIgD⁻ CD38^(hi) plasma cells were sorted and the rearranged Ig heavychain V regions (V_(H)) from genomic DNA of single cells was amplifiedand directly sequenced as previously described (Sims et al., Blood, inpress, 2005). The V_(H) gene sequence was compared to the closestgermline gene and the number of mismatches was determined. The frequencyof mismatches for stimulated cord blood B cells was compared withpurified cord blood B cells that were analyzed before culture.

Example 2 Induction of Apoptosis

Consistent with a pro-apoptotic effect, IL-21-induced DNA fragmentationas determined by TUNEL staining of B220⁺B cells stimulated withanti-CD40 (FIG. 1A, panels ii versus i), anti-IgM+IL-4 (panels iv versusiii), or LPS (panels vi versus v). Interestingly, IL-21 could induceapoptosis of anti-CD40 stimulated B cells, even though it augmented³H-thymidine incorporation in these cells.

IL-21 induced apoptosis can be inhibited by caspase inhibitors (Mehta etal., J. Immunol. 170:4111-18, 2003) and correspondingly, IL-21 inducedcleavage of PARP, a caspase substrate, either alone (FIG. 1B, panel i)or when combined with LPS (panel ii) or anti-CD40 (panel iii). AlthoughBcl-2 mRNA was observed to decrease after treatment with IL-21 (Mehta etal., J. Immunol. 170:4111-18, 2003), no change was observed in Bcl-2protein levels (FIG. 1C). In addition to its pro-apoptotic effect on Bcells stimulated with mitogens, IL-21 also induced apoptosis of freshlyisolated naïve splenic B cells within three days of culture. Conversely,it did not diminish the number of CD3⁺ T cells, and in fact increasedthe survival of both CD4⁺ and CD8⁺ T cells, with a greater effect onCD8⁺ T cells (FIG. 1D).

The pro-apoptotic effects of IL-21 on B cells in vitro were surprisinggiven that IL-21R expression is essential for normal Ig production invivo (Ozaki et al., Science 298:1630, 2002). To help explain thisapparent paradox, IL-21 transgenic (TG) mice were generated using avector (Pircher et al., EMBO J. 8:719, 1989; Held et al., J. Exp. Med.184:2037, 1996) that drives expression in T, B, and NK cells. Foundermice expressing murine IL-21 uniformly exhibited growth retardation anddied before sexual maturity. Thus, TG mice expressing human IL-21 weregenerated. Human IL-21 can stimulate murine cells in vitro but is likelyto bind the murine IL-21R with lower affinity. Four founders of thehuman IL-21 TG mice also exhibited growth retardation and died beforeadulthood, but three viable lines were obtained (FIG. 2A). The line withthe greatest IL-21 expression was lost, consistent with toxicityresulting from high expression of IL-21. All studies were performed onthe remaining lines (#5 and #7).

In the normal mouse spleen, approximately 90% of B cells are maturefollicular (FO) or marginal zone (MZ) cells (Martin and Kearney, Nat.Rev. Immunol. 2:323, 2002). Immature or newly formed (NF) B cells areproduced in the bone marrow and migrate to the spleen as transitional T1cells where they mature into T2 cells. Only 1-3% of these cellsdifferentiate into mature B cells (Hao and Rajewsky, J. Exp. Med.194:1151, 2001; Loder et al., J. Exp. Med. 190:75, 1999), which candevelop into memory B cells and antibody forming plasma cells afterantigen stimulation (Sze et al., J. Exp. Med. 192:813, 2000; Calame,Nat. Immunol. 2:1103, 2001). Staining of splenocytes from the humanIL-21 transgenic mice with antibodies to IgM and CD21 (marked as “M” inFIG. 2B, panel i) and T2/MZ B cell populations were decreased, whereasT1 cells appeared to be intact or increased (FIG. 2B, panel ii versusi). Consistent with this, the percentage of CD21^(int)CD23^(high)“mature” follicular (FO) B cells (FIG. 2B, panel iii) was markedlydecreased in transgenic mice (panel iv), while “immature” newly formed(NF) CD21^(low)CD23^(low) B cells (panel iii) were dramaticallyincreased in the transgenic mice (panel iv), suggesting either augmentedproduction or decreased death of these cells or a block in theirdifferentiation to mature B cells. Similarly, an apparent increase inimmature B cells (compare FIG. 2C to 2B, panel ii versus i and iv versusiii) was observed in mice injected with IL-21 DNA usinghydrodynamic-based transfection (Liu et al., Gene Ther. 6:1258, 1999;Zhang et al., Human Gene Ther. 10:1735, 1999). IL-21 expression vectorDNA was injected into wild type mice allowing examination of the effectof a relatively acute increase in murine IL-21 in wild type mice, againfinding

Consistent with this increase in “immature” B cells, staining with AA4.1mAb, which binds to a 130-140 kDa marker of immature B cells (Allman etal., J. Immunol. 167:6834, 2001), also was increased in IL-21 transgenicmice (FIG. 3A, panel ii versus i) and mice injected with IL-21 DNA.Analysis of immature and mature populations as defined by AA4.1/B220staining revealed an increase in the T1:T2 transitional cell ratio(i.e., CD23^(low):CD23^(high) ratio) in the AA4.1^(high)B220⁺ cells inthe transgenic mice (panel iv versus iii) and mice injected with IL-21DNA. However, the mature AA4.1^(low)B220⁺ cells showed a decrease in therelative number of CD23^(high) cells as well as a corresponding increasein CD23^(low) cells (panel vi versus v), which was inconsistent with theconclusions of changes in FO and MZ B cells based on CD21/IgM andCD21/CD23 staining in FIGS. 2B and 2C. IL-21 potently decreased CD23expression on naive B cells and also on B cells stimulated with LPS oranti-CD40 (FIG. 3B), and modestly diminished expression of CD21. Thus,the apparent decrease in FO cells induced by IL-21 (FIGS. 2B and 2C,panels iii and iv) could have at least in part reflected anIL-21-mediated decrease in expression of CD23. Thus, surface expressionof IgM and IgD was evaluated. These are not affected by IL-21. Among theAA4.1^(high) immature cells, the IgM/IgD staining pattern revealed thatthe T1:T2 (IgM⁺IgD^(low):IgM^(high)IgD⁺) ratio was not increased, andwas, if anything, somewhat decreased (FIG. 3A, panel viii versus vii).The AA4.1^(low) splenic population exhibited a modest decrease in themost mature FO B cell subset (IgD^(high)IgM^(low)) in both IL-21 TG mice(FIG. 3A, panel x versus ix, upper left quadrant) and mice injected withIL-21 DNA.

In contrast to the CD21/CD23 result, the changes in IgD^(low)IgM^(high)MZ cells in either the TG (lower right quadrant) or the injected micewere modest. Interestingly, in both IL-21 TG mice (lower left quadrant)and IL-21-injected mice, there was a dramatic increase in theIgD^(low)IgM^(low) population of AA4.1^(low) splenocytes, whichrepresents cells that have undergone Ig class switch recombination.Overall, based on the AA4.1 and IgM/IgD staining, IL-21 increased thetotal number of splenocytes and total B cells, with a marked increase inthe number of immature AA4.1^(high) B cells (FIG. 3C). AA4.1^(low)cells, which include both mature B cells and post-switch cells, werenormal in number (FIG. 3C). However, of these cells, mature B cells werediminished based on the decrease in IgD⁺ cells in FIG. 3A (panel xversus ix, see upper right quadrant), whereas post-switch cells (IgD⁻)were markedly increased (FIG. 3A, panel x versus ix, lower leftquadrant). Thus, IL-21 stimulation decreases the number of mature Bcells, but increases immature cells and drives the differentiation ofpost-switch cells.

Example 3 Effect of IL-21 on Splenic Architecture

The effect of IL-21 on splenic follicular architecture was evaluated.Immunostaining with antibodies to IgM, IgD, MAdCAM-1, and MARCO showedthat spleens from IL-21 transgenic mice had intact FO and MZ structures(FIGS. 4D-F versus FIGS. 4A-C). In contrast, spleens from mice injectedwith IL-21 DNA exhibited a loss of MZ B cells as revealed by the lack ofa bright red ring of IgM^(high)IgD⁻ B cells around the IgM⁺IgD⁺ “green”follicles (FIG. 4G versus FIG. 4A). Nevertheless, the MAdCAM-1⁺ marginalsinus (FIG. 4H versus FIG. 4B) and the MARCO⁺ MZ macrophages (FIG. 41versus FIG. 4C) were still present in these mice, indicating a loss of Bcells from this region rather than a loss of the MZ structure. Thefollicular dendritic cell, CD4⁺, and CD8⁺ areas were normal in bothIL-21 injected and transgenic mice. Thus, overall, the results revealedthat chronic human IL-21 signaling in the TG mice did not affect theoverall MZ structure, but led to increased immature B cells andaccumulation of Ig class-switched B cells. Exposure to the more acutelevels of murine IL-21 in the injected mice led to similar changes insplenic B cell populations, including that MZ B cells are retained asevaluated by CD1d and CD9, but these cells had apparently migrated outof the MZ (FIG. 4G versus FIG. 4A).

To evaluate the contribution of apoptosis to the effects of IL-21,annexin V staining was examined. Immunostaining with antibodies to IgM,IgD, MAdCAM-1, and MARCO showed that spleens from IL-21 transgenic micehad intact FO and MZ structures (FIGS. 4D-F versus FIGS. 4A-C). Incontrast, spleens from mice injected with IL-21 DNA exhibited a loss ofMZ B cells as revealed by the lack of a bright red ring ofIgM^(high)IgD⁻ B cells around the IgM⁺IgD⁺ “green” follicles (FIG. 4Gversus FIG. 4A). Nevertheless, the MAdCAM-1⁺ marginal sinus (FIG. 4Hversus FIG. 4B) and the MARCO⁺ MZ macrophages (FIG. 41 versus FIG. 4C)were still present in these mice, indicating a loss of B cells from thisregion rather than a loss of the MZ structure. The follicular dendriticcell, CD4⁺, and CD8⁺ areas were normal in both IL-21 injected andtransgenic mice. Thus, overall, the results revealed that chronic humanIL-21 signaling in the TG mice did not affect the overall MZ structure,but led to increased immature B cells and accumulation of Igclass-switched B cells. Exposure to the more acute levels of murineIL-21 in the injected mice led to similar changes in splenic B cellpopulations, including that MZ B cells are retained as evaluated by CD1dand CD9, but these cells had apparently migrated out of the MZ (FIG. 4Gversus FIG. 4A).

To evaluate the contribution of apoptosis to the effects of IL-21,annexin V staining was examined. Higher annexin V staining of B220⁺ Bcells was seen in IL-21 TG mice than in WT littermates (FIG. 5A, paneli), and similarly, injection of mice with the IL-21 plasmid increasedannexin V staining (FIG. 5A, panel ii). Thus, the reduction in mature FOB cells was, at least in part, caused by apoptotic death in vivo.

In contrast to the pro-apoptotic effect of IL-21 on mature B cells,IL-21 did not induce death of CD3⁺ T cells, and in fact was potentlyanti-apoptotic for CD8⁺ T cells (FIG. 1D). Consistent with this, IL-21transgenic mice had a markedly decreased CD4/CD8 T cell ratio in thymus,spleen, and lymph nodes (FIG. 5B, panel ii versus i), resulting from aselective increase in CD8⁺ T cells. Injection of the IL-21 plasmid in WTmice also decreased the CD4/CD8 splenic T cell ratio (FIG. 5B panel ivversus iii), with a marked increase in CD8⁺ T cells at day 7(approximately 41 million CD8⁺ splenic T cells in IL-21 injected versus13 million in the control). The ability of IL-21 to expand CD8⁺ T cellsin vivo could represent a redundant, rather than distinctive effect ofIL-21, as IL-21R^(−/−) mice do not exhibit an increased CD4/CD8 T cellratio (Ozaki et al., Science 298:1630-1634, 2002), presumably becausesignaling in response to other γ_(c)-dependent cytokines that areimportant for CD8⁺ T cell homeostasis, such as IL-7 and IL-15 (Schlunset al., Nat Rev Immunol. 3:269-279, 2003), is still intact. The dataestablish that IL-21 is a third γ_(c)-dependent cytokine thatcontributes to CD8⁺ T cell homeostasis.

Although IL-21 increased B cell apoptosis in vivo, it also clearlyinduced an increase in Ig class-switched B cells (see above and FIG.3A). Indeed, IL-21 TG mice had increased levels of serum IgG and IgM(FIG. 5C), and increased surface IgG1⁺ B cells in the spleen (FIG. 5D).Moreover, mice injected with IL-21 DNA exhibited increased IgG⁺ B cellsby immunohistology. Thus, the effects of IL-21 on the primary antibodyresponse to ovalbumin, a T cell dependent antigen, were analyzed. Theconcentrations of antigen-specific IgM and IgG were similar in WT and TGmice (FIG. 5E), demonstrating that “extra” IL-21 did not interfere witheffective Ig production and thus was not apoptotic for the Ig producingcells. Thus, the data collectively indicate that IL-21 promotesmaturation of B cells, accompanied by class switching and plasma cellformation.

Example 4 Effect of IL-21 on B Cell Maturation

To determine whether these maturation effects resulted from directstimulation of B cells by IL-21, B cells cultured for 48 hours wereanalyzed in the presence of anti-IgM in combination with either IL-4,anti-CD40, or both stimuli (FIG. 6A) and IL-21. IL-21 stimulated B cellproliferation induced by anti-IgM, especially in the presence ofanti-CD40, but it inhibited proliferation to anti-IgM plus IL-4 (FIG.6A); however, costimulation of anti-IgM+IL-4-activated B cells withanti-CD40 restored the ability of IL-21 to augment proliferation (FIG.6A). Consistent with the effect of IL-21 on antibody production (FIG.5C), IL-21-induced expression of syndecan-1 (CD138), a plasma cellmarker (FIG. 6B), and surface IgG1 (FIG. 6C) (see panels ii versus i andiv versus iii) in B cells stimulated with anti-IgM with or without IL-4.The surface IgG1⁺ cells shown in FIG. 6C did not all express Syndecan-1,indicating that IL21 was increasing memory cells as well as plasmacells. Thus, culture conditions that allow IL-21 apoptotic effects alsoallow IL-21 induced B cell differentiation. Overall, IL-21 haspro-apoptotic effects for mature FO B cells, induces an increase inimmature B cells, alters the B cell phenotype, and is a potent inducerof B cell maturation to memory B/post-switch cells and plasma cells.IL-21 also induces differentiation of human B cells into plasma cells(FIG. 8) and memory B cells.

Example 5 Blimp-1 and Bcl-6

As noted above, IL-21 is pro-apoptotic based on a caspase dependentmechanism, but its role related to B cell maturation and plasma celldifferentiation was unclear. Blimp-1 is a transcription factor that hasbeen identified as a master regulator of plasma cell differentiation(Turner et al., Cell 77:297-306, 1994), whereas Bcl-6 and Pax5 arerequired for germinal center formation (reviewed in Calame et al., Annu.Rev. Immunol. 21:205-230, 2003). Interestingly, Blimp-1 and Bcl-6 havebeen shown to each inhibit expression of the other protein, and Blimp-1additionally is an inhibitor of the expression of Pax5 (Shaffer et al.,Immunity 17:51-62, 2002; Calame et al., Annu. Rev. Immunol. 21:205-230,2003).

Expression of these proteins was examined in Bcl-1 3B3 cells, a B celllymphoma cell line in which treatment with IL-2 and IL-5 can induceplasmacytic differentiation into immunoglobulin-secreting cells (Messikaet al., J. Exp. Med. 188: 515-525, 1998). The effect of IL-21 on thesecells was examined, and it was found that IL-21 induced expression ofsyndecan-1, while it decreased expression of MHC class II, consistentwith plasma cell differentiation and Blimp-1 induction (Turner et al.,Cell 77:297-306, 1994; Piskurich et al., Nature Immunol. 1:526-532,2000) (FIG. 7A). Consistent with the effect of IL21 on CD23 expressionin splenic B cells (FIG. 3B), IL-21 also decreased CD23 expression inBcl-1 cells (FIG. 7A).

Correlation of these findings with the expression of Blimp-1, Bcl-6 andPax5 was sought. Based on real-time PCR analysis, IL-21 inducedexpression of mRNA for both Blimp-1 and Bcl-6, whereas it inhibitedexpression of Pax5 mRNA (FIG. 7B). The induction of Blimp-1 was at leastas potent as that seen with the combination of IL-2 and IL-5; asexpected, the combination of IL-2 and IL-5 inhibited expression ofBcl-6. The induction of both Blimp-1 and Bcl-6 was further confirmed byexamining B cells purified from the spleen. Induction was not seen incells treated with anti-IgM alone, but the addition of IL-21 inducedBlimp-1 protein expression (FIG. 7C) as well as Blimp-1 and Bcl-6 DNAbinding activity as evaluated by electrophoretic mobility shift assays(FIG. 7D).

IL-21 is the only type I cytokine that can induce apoptosis of restingnaïve B cells, whereas other γ_(c) family cytokines are typicallyanti-apoptotic. In the context of antigen activation, IL-2 can promote Tcell death via a process known as activation-induced cell death (AICD)(Lenardo et al., Annu. Rev. Immunol. 17:221, 1999). However, IL-21 isdifferent in that it is pro-apoptotic for B and NK cells instead of Tcells (Kasaian et al., Immunity 16:559, 2002), and whereas AICD requiresa prior activation signal, IL-21-induced apoptosis does not. In additionto apoptotic effects on mature B cells, IL-21 induced the accumulationof transitional B cells in the periphery. This could reflect homeostaticcompensation for the reduction in peripheral mature B cells, the abilityof IL-21 to promote maturation and/or survival of immature B cells fromthe bone marrow, and/or an ability of IL-21 to interfere with signalsthat promote differentiation from transitional to mature B cells.Strikingly, in mice injected with the IL-21 plasmid, the MZ was depletedof B cells, but the marginal sinus and associated MZ macrophages wereleft intact. It is possible that the MZ B cells migrated out of the MZ,as is found following exposure to particulate antigens such as bacteria(Martin et al., Immunity 14:617, 2001), where MZ B cells migrate to theT-B border, followed by the accumulation of Ag-specific plasmablasts inthe red pulp. The extrafollicular foci of plasma cells are expanded inIL-21-treated mice and this might reflect this pattern of migration anddifferentiation. The MZ B cell population appeared intact in IL-21 TGmice.

IL-21 TG mice exhibited elevated serum IgM and IgG and had increasedsurface IgG1⁺ B cells in the spleen, suggesting that IL-21 promoted Igisotype switching in vivo. These mice could mount productive primary Igresponses (involving all isotypes) to a T cell dependent antigen. IL-21downregulated CD23 expression on mature B cells and promoteddifferentiation to Ig secreting plasma cells both in vivo and in vitro,whereas IL-4 inhibits plasma cell differentiation (Knodel et al., Eur.J. Immunol. 31:1972-1980, 2001) and induces CD23 expression on mature Bcells (Nelms et al., Annu. Rev. Immunol. 17:701-738, 1999).Interestingly, accumulation of surface IgG1⁺ cells in response to IL-21is reduced in the presence of IL-4, whereas the induction of syndecan-1⁺cells was not altered. Thus, some but not all of the effects of IL-21 onthe B cell immune response can be modulated by IL-4. The observationthat IL-21 has anti-apoptotic effects on some myeloma cell lines (Brenneet al., Blood 99:3756, 2002) is consistent with the observationspresented herein that IL-21 is an inducing factor for plasma cells. Itseffect on plasma cell differentiation could result from induction ofBlimp-1, whereas the induction of Bcl-6 is likely to be important forsubsequent differentiation of germinal center cells into memory cells.It is noteworthy that IL-21 induced both Blimp-1 and Bcl-6, consideringthat these are usually mutually antagonistic (Shaffer et al., Immunity17:51-62, 2002; Calame et al., Annu Rev Immunol. 21:205-230, 2003). Thisis another indication of the overall potency of IL-21 in stimulatingvarious aspects of the differentiation of B cells, and havingdifferential effects in a context-regulated manner.

IL-21 has pleiotropic effects on lymphoid lineages. Whereas IL21 ispro-apoptotic for mature B cells, and anti-apoptotic actions for Tcells, IL-21 has complex actions for B cells, as demonstrated herein.The degree of IL-21-induced apoptosis depends on the nature of othersignals to the B cell. Early in the B cell response, IL-21 can functionas an apoptotic signal. When bystander B cells are stimulated in anantigen-non-specific manner by activated T cells mediated by CD40engagement, but in the absence of B cell receptor signaling, IL-21 leadsto apoptosis. Following the initiation of a B cell immune response,IL-21 can eliminate “bystander” B cells responsible for the non-specifichypergammaglobulinemia that is initially observed.

However, in B cells fully activated by B cell receptor signaling and anappropriate T cell signal (e.g., CD40L) (Lee et al., Proc. Natl. Acad.Sci. USA 96:9136-9141, 1999), IL-21 enhances Ig production, isotypeswitching, and memory cell and plasma cell production. In this regard itis demonstrated herein that IL-21 upregulates both Blimp-1 and Bcl-6.Moreover, it is unprecedented that both Blimp-1 and Bcl-6 would areinduced. Thus, IL-21 is a major regulator of the B cell immune response.This finding explains how IL-21 can drive differentiation of B cellsboth into post-switch memory cells as well as to plasma cells,establishing IL-21 as a global regulator of B cell differentiation andfunction.

Example 6 IL-21 Induces Human Plasma Cell Differentiation

The capacity of IL-21 to co-stimulate responses of purified human Bcells was evaluated. Plasma cells were identified phenotypically asCD19^(lo/+)IgD⁻ CD38^(hi) cells. Freshly obtained B cells or thoseretrieved from cultures were stained with anti-CD19, anti-IgD andanti-CD38. CD19⁺ cells were assessed for expression of IgD and CD38.Staphylococcus aureus Cowan I (SAC) is a polyclonal B cell activatorthat induces rapid activation, differentiation and Ig production of Bcells in the presence of IL-2. However, in the absence of IL-2, littleplasma cell differentiation occurs. In contrast, costimulation ofpurified peripheral blood B cells with both SAC and IL-21 resulted inmarked down-modulation of IgD and substantial differentiation of plasmacells that were phenotypically identified as CD19lo/+IgD−CD38hi cells.

We next examined whether IL-21 would co-stimulate B cells activatedspecifically through the B cell receptor and/or CD40. As shown in FIG.9A, only a small fraction (1.3%) of freshly isolated human peripheral Bcells are IgD⁻CD38^(hi) plasma cells; the majority are IgD⁺CD38^(lo/Int) naïve B cells and a smaller percentage are IgD⁻CD38^(−/ol) post-switched memory B cells. In the presence of anti-CD40(which can stimulate both naïve and memory B cells), IL-21 inducedmaximal proliferation of B cells, whereas IL-2 had little effect (FIG.9B). In contrast, in the presence of anti-IgM (which stimulatesIgD⁺/IgM⁺B cells only), IL-21 induced only minimal proliferation.Notably, IL-2 enhanced the proliferation of B cells stimulated withanti-IgM and IL-21, whereas it had little effect on B cells stimulatedwith anti-CD40 and IL-21. When B cells were triggered through both CD40and IgM, IL-21 induced a proliferative response that was comparable tothat noted with anti-CD40 and IL-21. As with the anti-IgM and IL-21costimulation, IL-2 also increased the magnitude of the response whencells were stimulated with the combination of IL-21, anti-IgM andanti-CD40.

Flow cytometric evaluation was carried out to assess the impact of IL-21on human B cells. When negatively selected peripheral B cells werecultured with cytokines alone, modest changes in B cell phenotype werenoted as determined by IgD and CD38 expression as shown in FIG. 9C(panels a-d). However, IL-21 in the absence of any other stimulusincreased the percentage of IgD⁻ CD38^(hi) plasma cells modestly andthis effect was augmented by IL-2 (FIG. 9C panels c and d). The abilityof IL-21 to induce moderate plasma cell differentiation was alsoobserved with anti-IgM-stimulated B cells, and again there was anincrease in the presence of IL-2 (FIG. 9C panels g and h). In addition,IL-21 induced a striking loss in the numbers of B cells in anti-IgMstimulated cultures (FIG. 9C panels g and e), that was largely reversedby the addition of IL-2 (FIG. 9C panels h and g). Moreover, IL-21 or thecombination of IL-2 and IL-21 induced marked down-regulation of surfaceIgD by anti-IgM stimulated B cells (FIG. 9C, panels g and h) and thiswas noted by day 4 of culture. IL-21 also induced IgD down-modulation byanti-CD40-stimulated B cells, as well as a dramatic increase in cellularexpansion and plasma cell differentiation that was observed as early asday 4 of culture (FIG. 9C, panels i-1). T cell-dependent B cellresponses involve engagement of both surface Ig as well as CD40 by CD154expressed by activated T cells (Defrance et al., J. Exp. Med.175:671-682, 1992).

The impact of IL-21 on B cells stimulated through both the B cellreceptor (BCR) and CD40 was therefore examined. The combination ofanti-IgM and anti-CD40 in the presence of IL-21 resulted indown-modulation of IgD by nearly all B cells (FIG. 9C panels o and m).Moreover, the largest percentage of plasma cells was generated when bothsurface IgM and CD40 were engaged and the cells were co-stimulated withIL-21 in the presence or absence of IL-2 (FIG. 9C panels o and p).Evaluation of CFSE dilution revealed that IL-21 induced thedifferentiation of plasma cells from a dividing B cell precursor thathad diluted CFSE with or without costimulation (FIG. 9D). Numericcalculation in repetitive experiments demonstrated the significantincrease in both the percentage (FIG. 10A) and absolute cell number(FIG. 10B) of plasma cells in cultures co-stimulated with IL-21. Themajority of the IL-21-driven plasma cells expressed conventional plasmacell antigens and were CD19^(lo), CD20^(lo), CD22^(lo), CD21^(lo), IgD⁻,CD38^(hi), BCMA⁺, IL-6R⁺ and CD27^(hi).

CD27 expression indicates that human B cells have somatically mutated Iggenes, and therefore are considered to be memory B cells. These includeboth IgD⁺CD27⁺ B cells as well as IgD⁻CD27⁺ post-switched B cells. Toaddress whether IL-21 had the capability to drive plasma celldifferentiation from both naïve and memory B cells, cord blood B cellswere used as a natural source of naïve CD27⁻IgD⁺B cells, and CD27⁺ (IgD⁻and IgD⁺) memory B cells were isolated from peripheral blood by cellsorting. As shown in FIGS. 10A and B, IL-21 co-stimulated considerableproliferation of both naïve cord blood and memory B cells. Although cordblood B cells co-stimulated with IL-21 proliferated less well than adultCD27⁺ memory B cells (FIGS. 11A and B), the capacity of IL-21 todown-modulate IgD and induce differentiation of plasma cells wasdramatic in both populations (FIGS. 11C and D). Nearly all survivingIL-21-stimulated cord blood B cells activated with either anti-CD40 oranti-IgM and anti-CD40 had down-modulated IgD and/or differentiated intoplasma cells. neither anti-CD40 nor anti-IgM and anti-CD40 stimulationof cord blood B cells alone led to the generation of any plasma cells inthe absence of IL-21 (FIG. 11C). However, IL-21 induced thedown-modulation of IgD on stimulated cord blood B cells within 3 days ofculture, and by day 6 many plasma cells were observed. In contrast,IL-21-induced differentiation of plasma cells from adult peripheralblood B cells could be observed within 4 days of culture.

IL-21 is a powerful co-stimulator of human plasma cell differentiationfrom human naïve as well as memory B cells. In contrast, a variety ofother cytokines, including IL-2, IL-4, IL-6, and IL-10, have minimalability to support the generation of plasma cells from comparablystimulated B cells. The combination of anti-IgM and anti-CD40, whichmost closely mimics B cell activation via antigen and T cellinteraction, is the most effective signal in promoting the maximaldifferentiation of plasma cells driven by IL-21. Isolated BCRcross-linking, which mimics the “antigen-only” signal, primes B cellsfor IL-21-mediated death. These results indicate that IL-21 is involvedin cell fate decisions by activated B cells and can function toeliminate B cells that have been activated by antigens or autoantigensin the absence of T cell signals.

Example 7 IL-21 Induces Ig Secretion From Human Naïve and Memory B Cells

Analysis of culture supernatants confirmed that IL-21 induced thesecretion of Ig. Unlike IL-2, or cultures with no added cytokines, IL-21co-stimulated considerable production of both IgG and IgM from totaladult peripheral B cells, as well as naïve cord blood B cells activatedwith anti-CD40 or anti-IgM and anti-CD40 (FIGS. 12A and B). The amountof Ig produced generally correlated with the frequency of plasma cellsin the cultures. IgM and IgG could be assayed in the culturesupernatants as early as day 4 of culture. IL-21 induced large amountsof IgG from both naïve CD27⁻ adult and cord blood B cells (FIGS. 12B andC), consistent with its capability to function as a switch recombinationfactor for human B cells. All IgG isotypes were produced from adultCD27⁺ memory B cells, as is expected from a population of post-switchedmemory B cells (FIG. 12C). Although all IgG isotypes were produced fromnaïve cord blood B cells stimulated with the combination of IL-2, IL-21,anti-CD40 or anti-IgM and anti-CD40, IgG3 predominated (FIG. 12C). Incontrast, both IgG1 and IgG3 were the dominant IgG isotypes produced byIL-21 co-stimulated CD27 adult naive B cells (FIG. 12C). In addition,stimulation with of IL-21 and anti-CD40 or IL-21, anti-CD40 and anti-IgMof peripheral blood B cells had no effect on IgE production, but didcostimulate IgA production without or with IL-2 (1-2 μg/ml vs. 4-6 μg/mlof IgA respectively).

Example 8 IL-21 Induces Expression of Blimp-1, AID and Bcl-6 in Human BCells

A variety of transcription factors are known to regulate specific stagesof B cell maturation. Therefore, it was determined whether IL-21costimulation upregulated expression of Blimp-1, which is essential forplasma cell differentiation (Turner et al., Cell 77:297-306, 1994), AID,which is involved in CSR (Durandy, Eur. J. Immunol. 33:2069-2073, 2003),Bcl-6, which is involved in germinal center reactions (Ye et al., Nat.Genet. 16:161-170, 1997), or PAX-5, which is required for the generationof B cells (Rolink et al., Immunol. Rev. 187:87-95, 2002). IL-21 inducedexpression of both Blimp-1 (FIG. 13A panel a) and AID (FIG. 13A panel b)mRNA. Blimp-1 was induced by IL-21 co-stimulated B cells activated withanti-CD40 or anti-IgM and anti-CD40, and less so with anti-IgM only(FIG. 13A panel a). However, in the presence of IL-2, IL-21 also inducedBlimp-1 expression without additional costimulation. Furthermore, IL-2enhanced the IL-21-mediated Blimp-1 expression in B cells activated byanti-CD40 or anti-CD40 and anti-IgM (FIG. 13A panel a). AID mRNA wasinduced by IL-21 only when B cells were activated with anti-CD40 or bothanti-IgM and anti-CD40, and was enhanced by the presence of IL-2,especially when B cells were stimulated with anti-IgM (FIG. 13A panelb). Bcl-6 mRNA was induced to a lesser degree by IL-21 (FIG. 13A panelc), whereas IL-21 had little effect on PAX-5 mRNA (FIG. 14A panel d). Todetermine whether IL-21 had the ability to induce these transcriptionfactors in naïve B cells, experiments using cord blood B cells wereundertaken. IL-21 co-stimulated a significant increase in Blimp-1 mRNAin naïve cord blood B cells, whereas the combination of IL-2 and IL-4exhibited no such activity (FIG. 13B). IL-21 also co-stimulated AID mRNAexpression, but no more effectively then the combination of IL-2 andIL-4 (FIG. 13B). In contrast, IL-21 had little effect on Bcl-6 or PAX-5mRNA levels.

IL-21 costimulation increased AID mRNA levels, differentiation of IgD⁺and IgD⁻ B cells, class switch recombination, generation of plasmacells, and Ig production, but it could not induce somatic hypermutation.No increase in mutation of V_(H) genes was detected in IgD⁻ CD38^(+/−) Bcells or IgD⁻ CD38^(hi) plasma cells isolated from cultures of cordblood B cells stimulated with anti-CD40 with or without anti-IgM in thepresence of IL-2 and IL-21 (FIG. 13C).

Blimp-1 is a transcriptional repressor that is necessary/sufficient forplasma cell differentiation in the mouse, and in human B cell lines(Turner et al., Cell 77:297-306; Shapiro-Shelef et al., Immunity19:607-620, 2003; Shaffer et al., Immunity 17:51-62, 2002). Consistentwith this, IL-21-driven plasma cell differentiation from both naive cordblood B cells as well as CD27+memory B cells was preceded by theinduction of Blimp-1 up-regulation. Other cytokines examined in thisstudy did not induce Blimp-1 expression, although IL-2 enhanced theeffects of IL-21. As indicated in Example 5, IL-21 induced Blimp-1expression in murine splenic B cells. The capacity of IL-21 toup-regulate Blimp-1 explains its ability to drive plasma celldifferentiation. In addition, IL-21 costimulation induced AID expressionwhich is normally down-modulated by Blimp-1 (Shaffer et al., Immunity17:51-62, 2002). Although, AID is a B cell-specific factor that issufficient for both CSR and SHM (Honjo et al., Immunity 20:659-668,2004), IL-21 costimulation induced CSR, but not SHM. Similar findingshave been reported with CD27—B cells stimulated with the combination ofSAC, IL-2, IL-10, and cross-linked anti-CD40 (Nagumo et al., Blood99:567-575, 2002). IL-4 and CD40 stimulation induced AID in cord blood Bcells, but neither CSR nor plasma cell differentiation. Previously, AIDhas been shown to be induced by IL-4, anti-CD40 or the combination ofboth IL-4 and anti-CD40 in adult B cells (Pene et al., J. Immunol.172:5154-5157, 2004; Zhou et al., J. Immunol. 170:1887-1893; Dedeoglu etal., Intl. Immunol. 16:395404, 2004). Although AID was induced tocomparable levels by IL-4 and IL-21, although only the latter inducedCSR. In this regard, engagement of CD40 induced both AID and SHM inhuman Ramos B cell lines (Zan et al., Immunity 18:727-738, 2003; Failiet al., Nat. Immunol. 3:815-821, 2002). These results indicate thatup-regulation of AID has different effects on B cells at differentstages of differentiation. Moreover, our data indicate that theinduction of AID mRNA is not sufficient to induce CSR or SHM in allcircumstances. Without being bound by theory, it is likely that IL-21can induce CSR, the activity of AID that is governed by the C-terminalportion of the molecule and involves the capacity to bind DNA-PKcs Wu etal., J. Immunol. 174:934-941, 2005), whereas it is not able to induceSHM that is regulated by the N-terminal of the protein by unknownmolecules (Shinkura et al., Nat. Immunol. 7:707-712, 2004).

Example 9 Effect of Cytokines on Plasma Cell Generation Promoted byIL-21

The effects of IL-21 can be modulated by other cytokines. As shown inFIG. 14A, IL-4 partially inhibited the down-modulation of surface IgDinduced by IL-21 in cells stimulated with Staphylococcus aureus Cowan I(SAC), which is a polyclonal B cell activator that induces rapidactivation, differentiation and Ig production of purified B cells in thepresence of IL-2 (Teranishi et al., J. Immunol. 133:3062-3067, 1984)(FIG. 14A panels e and f). Moreover, IL-4 also inhibited IgDdown-modulation induced by IL-21 and anti-IgM or the combination ofanti-IgM and anti-CD40 (FIG. 15A panels h, i, n and o), but not byanti-CD40 alone (FIG. 14A panels l and k). Importantly, IL-21-inducedgeneration of plasma cells was also inhibited by IL-4 in culturesco-stimulated by SAC (FIG. 14A panels e and f), or in culturescontaining the combination of IL-21, anti-IgM and anti-CD40 (FIG. 14Apanels n and o) but not in cultures of IL-21 and anti-CD40 (FIG. 14Apanels k and i). This was reflected in the quantity of secreted Ig (FIG.14B), as well as the levels of AID or Blimp-1 mRNA (FIG. 14C). Theeffect on AID mRNA was only observed in cultures stimulated by anti-IgMand anti-CD40. The addition of IL-2 did not rescue IgG production inIL-4 suppressed cultures (FIG. 14B), but did allow for thedifferentiation of IgM-producing plasma cells.

The next experiments compared the impact of IL-21 with IL-0 which isknown to support plasma cell differentiation (Briere et al., J. Exp.Med. 179:757-762, 1994; Rousset et al., Proc. Natl. Acad. of Sci. USA89:1890-1893, 1992). IL-10 induced minimal B cell responses compared toIL-21 when added in the same culture conditions (FIG. 15A-). IL-10co-stimulated neither Ig-production (FIG. 15A) nor differentiation (FIG.15B) of naïve cord blood B cells under any condition, but did induce lowlevels of Ig and small numbers of cells to differentiate into plasmacells upon culture of purified CD27⁺ memory B with anti-CD40 or anti-IgMand anti-CD40. Moreover, IL-10 had no effect on B cell responsessupported by IL-21 in contrast to the effects of IL-4.

The effect of IL-21 was then compared to that of the combination of IL-2and IL-10, which has been shown to support plasma cell differentiationunder some circumstances. As shown in FIG. 16, stimulation of purifiedperipheral blood B cells with anti-CD40 and the combination of IL-2 andIL-10 induced proliferation (FIG. 16A), as well as the differentiationof plasma cells (FIG. 16B) and IgG secretion (FIG. 16C), although theresponses were not as robust as those induced by anti-CD40 and IL-21. Incontrast, stimulation of naïve cord blood B cells with anti-CD40 and thecombination of IL-2 and IL-10 failed to stimulate any of theseresponses, although IL-21 induced proliferation, plasma cell generationand IgG secretion from anti-CD40-stimulated cord blood B cells (FIG.16A-C). Notably, however, the combination of IL-2 and IL-10 inducedupregulation of both BLIMP-1 and AID mRNA, although AID mRNA was inducedsubstantially more effectively by IL-21 (FIG. 16D).

IL-2 has previously been shown to drive plasma cell differentiation whenB cells are activated with SAC (Teranishi et al., J. Immunol.133:3062-3067, 1984), as well as co-stimulate plasma celldifferentiation in the presence of anti-CD40 and IL-10 (Arpin et al., J.Exp. Med. 186:931-940, 1997; Splawski et al., Eur. J. Immunol.28:4248-4256, 1998) or IL-6 (Jego et al., Blood 97:1817-1822, 2001;Vernino et al., J. Immunol. 148:404410, 1992). IL-4, on the other hand,has been shown to both promote and inhibit B cell responsiveness tovarious stimuli (Arpin et al., J. Exp. Med. 186:931-940, 1997; Splawskiet al., J. Immunol. 150:1276-1285, 1993; Jelinek et al., J. Immunol.141:164-173, 1988; Fujieda et al., J. Immunol. 155:2318-2328, 1995;Splawski et al., J. Immunol. 142:1569-1575, 1989; Jabara et al., J. Exp.Med. 172:1861-1864, 1990). IL-2 enhanced the effect of IL-21 on plasmacell differentiation induced by the combination of anti-IgM andanti-CD40, whereas IL-4 inhibited it. Moreover, IL-4 blockedIL-21-induced death of naive B cells stimulated with anti-IgM. Theseresults indicate an antagonistic effect of IL-4 on the actions of IL-21in B cells stimulated by anti-IgM or the combination of anti-IgM andanti-CD40. Antagonism between IL-4 and IL-21 was not consistentlyobserved when cells were stimulated with anti-CD40 alone, indicatingthat the nature of the stimulus influences the ability of B cells torespond to the combination of IL-4 and IL-21. Consistent with this, IL-4has been shown to enhance Ig production by IL-21 and anti-CD40 activatedCD27⁺ human splenic memory B cells (Pene et al., J. Immunol.172:5154-5157, 2004). This finding makes it unlikely that receptorcompetition can explain these results, owing to the fact that both IL-21and IL-4 utilize the γ_(C) for signaling (Zhang et al., Biochem.Biophys. Res. Comms. 300:291-296, 2003). In addition, IL-2, which alsosignals through a receptor containing γ_(C), reversed the IL-4mediated-inhibition of IgM-producing plasma cell generation, making theidea of receptor competition more remote. Furthermore, IL-2 synergizedwith IL-21 in fostering growth of anti-IgM and anti-CD40 stimulated Bcells. Thus, stimulus dependent inhibition of IL-21 responses by IL-4relates to the down stream signals generated by engagement of the IL-4receptor. In this regard, IL-4 is known to co-stimulate IgE synthesis byhuman B cells (Jabara et al., J. Exp. Med. 172:1861-1864, 1990), andalso induce the expression of CD23 on mature B cells (Nelms et al., Ann.Rev. Immunol. 17:701-738, 1999). However, IL-4 also has been reported torepress human B cells responses (Jelinek et al., J. Immunol.141:164-173, 1988; Splawski et al., J. Immunol. 142:1569-1575, 1989),and inhibit Blimp-1 induction as well as plasma cell differentiation inmice (Knodel et al., Eur. J. Immunol. 31:1972-1980, 2001). With theappropriate costimulation, IL-4 repressed Blimp-1 expression, andinhibited plasma cell differentiation, whereas IL-21 induced bothBlimp-1 and plasma cell differentiation. In the appropriate signalingcontext, the actions of the two cytokines appear to be mutuallyinhibitory of one another. In this regard, IL-21 has been reported toinhibit proliferation of both murine and human B cells stimulated withanti-IgM and IL-4 (Parrish-Novak et al., Nature 408:57-63, 2000; Ozakiet al., Science 298:1630-1634, 2002; Ozaki et al., J. Immunol.173:5361-5371, 2004) and to inhibit IL-4 induced IgE transcription (Sutoet al., Blood 100:45654573, 2002).

As discussed above, IL-4 repressed IL-21-driven plasma celldifferentiation following either SAC or anti-IgM and anti-CD40stimulation. IL-4R ligation induces primarily STAT6, which is essentialfor IL-4 signaling, including IL-4-induced up-regulation of AID in bothmice and humans (Zhou et al., J. Immunol. 170:1887-1893, 2003; Dedeogluet al., Intl. Immunol. 16:395404, 2004), whereas IL-21R engagementactivates primarily STAT1 and 3 and more weakly 5 (Ozaki et al., Proc.Natl. Acad. Sci. 97:11439-11444, 2000; Habib et al., Biochem.41:8725-8731, 2002; Habib et al., J Allergy & Clin. Immunol.112:1033-1045, 2003). The data suggest that IL-21 and IL-4 inhibit oneanother's actions after receptor engagement. Biologically, Tcell-dependent B cell responses may be regulated by T cells that eitherproduce IL-21, IL-4 or other cytokines at different times during animmune response or different subsets of T cells that express differentcytokine profiles. In support of this, IL-21 has been described to beproduced by a restricted subset of follicular T helper cells thatexpress CXCR5 in humans (Chtanova et al., J. Immunol. 173:68-78, 2004).

Example 10 IL-21-Induces Differentiation of Non-Dividing, TerminallyDifferentiated Plasma Cells

The status of IL-21-induced plasma cells was examined by cell surfacephenotype and cell cycle analysis. In addition to being CD19^(lo/+)IgD-CD38^(hi), the majority of IL-21, anti-CD40 and anti-IgM-inducedplasma cells were also found to express other typical plasma cellsmarkers such as IL-6R and BCMA (FIG. 17). Furthermore, when compared toIgD-CD38^(lo) cells, these plasma cells were CD40⁺, CD95^(lo),HLA-DR^(lo), CD9^(hi), CD63^(hi), CXCR4^(lo/+), CD44^(hi), CD49d^(lo),CD31^(hi) and CD62L^(hi) (FIG. 17). The majority of the IL-21-drivenplasma cells were also CD19^(lo), CD20^(lo), CD22^(lo), CD21^(lo) andCD27^(hi).

To evaluate whether IL-21-induced plasma cells were terminallydifferentiated nondividing cells, cell cycle analysis was also carriedout. To assess cell cycle position, B cells were stimulated with IL-21,anti-IgM and anti-CD40 and after 7 days of culture the resultant plasmacells were identified either as IgD-CD38^(hi) (FIG. 18B) orCD27^(hi)CD38^(hi) cells that were differentiated fromCD27^(lo)CD38^(lo) non-plasma cells (FIG. 18C). Both populations wereanalyzed for cell cycle progression by PI staining (FIG. 18D). As shownin FIG. 18D, the majority of IL-21-induced plasma cells as well asnon-plasma cells were not cycling as evidenced by the finding that theywere not in the S, G2 or M phase of the cell cycle, although 46% of thenon-plasma cells were found to be in S/G2/M on day 3, before plasma celldifferentiation.

Whether IL-21-induced plasma cells had differentiated to non-dividing Igsecreting cells, Ig production was examined in the presence of theinhibitor of proliferation, hydroxyurea. The addition of hyrdoxyurea atthe initiation of culture IL-21 drives human plasma cell differentiationresulted in a complete block of IL-21-induced proliferation and plasmacell differentiation (FIGS. 19A, B). However, when hydroxyurea was addedto the cultures at day 8 after differentiation of plasma cells,persistence of these cells was noted over the next three days of culture(FIG. 19B). The amount of Ig doubled during the last 3 days of culturewhich was not blocked by hydroxyurea, indicating that the ongoingproduction of Ig by plasma cells was not dependent on ongoingproliferation. These data demonstrate that the production of Ig byIL-21-induced plasma cells reflects secretory activity of terminallydifferentiated, non-dividing cells (FIG. 19C).

It will be apparent that the precise details of the methods orcompositions described can be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. A method of producing one or more of a memory B cell and a plasmacell, the method comprising: contacting a population of cells comprisingone or more of a mature B cell and a B cell progenitor with acomposition comprising IL-21, thereby inducing differentiation of atleast one of the mature B cell and the B cell progenitor into one ormore of a memory B cell and a plasma cell; and isolating or purifyingone or more of the memory B cell and the plasma cell.
 2. The method ofclaim 1, wherein the population of cells comprises bone marrow derivedcells, cord blood cells or peripheral blood cells.
 3. The method ofclaim 1, wherein the population of cells comprises a plurality ofisolated or purified cells, which isolated or purified cells compriseone or more of immature B cells and mature B cells.
 4. The method ofclaim 1, wherein the population of cells comprises human cells.